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Table of Contents
The Basics of PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 The Basics of PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 The Basics of RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Increasing RT-PCR Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolating High-Quality RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using RNase H– Reverse Transcriptases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Higher RT Incubation Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Additives to Enhance RT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RNase H Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improving Detection of Small Amounts of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . One-Step vs. Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improving RT-PCR Speciﬁcity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Priming cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Higher RT Incubation Temperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Minimizing Contaminating Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 4 5 5 5 6 6 7 7 7 7
RT-PCR Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5´ and 3´ RACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Quantifying mRNA Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Improving PCR Speciﬁcity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primer Annealing Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Touchdown PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primer Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primer Purity and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hot Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Magnesium Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Additives to Enhance PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nested PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Increasing PCR Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Template Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Template Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzyme Choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improving Fidelity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymes with Proofreading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzyme Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Things to Consider . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amplifying Long Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prevention of Carry-Over Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Puriﬁcation of PCR Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genotyping with Dinucleotide Repeat Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tools for Detecting Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 10 10 11 11 11 12 13 13 13 14 14 14 14 15 15 15 15 16 16 16 16 17 17 17 17
CHAPTER 10 CHAPTER 11 CHAPTER 12 APPENDIX A
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Selected Primer Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
C HA AP PT TE ER R 1 1 CH
The Basics of PCR and RT-PCR
Primer 1 Primer 2
The Basics of PCR he Polymerase Chain Reaction (PCR) process uses multiple cycles of template denaturation, primer annealing, and primer elongation to amplify DNA sequences (1). It is an exponential process since ampliﬁed products from the previous cycle serve as templates for the next cycle of
Component Template Final Concentration 104 –10 6 copies of DNA template 0.1– 0.5 µM 0.1– 0.5 µM 1X 1.0 – 3.0 mM 200 µM each dNTP 1– 4 units/100 µl reaction
10X Reaction buffer Magnesium dNTP mix Thermostable DNA polymerase
TABLE 1. Reaction components.
ampliﬁcation, making it a highly sensitive technique for the detection of nucleic acids. Typically, enough ampliﬁed product is generated after 20 to 30 cycles of PCR so that it can be visualized on an ethidium bromide-stained gel. The reaction is composed of several components (table 1). The template can include puriﬁed genomic or plasmid DNA; RNA converted to cDNA; or unpuriﬁed, crude biological samples such as bacterial colonies or phage plaques. The primers determine the sequence and the length of the ampliﬁed product. The most frequently used thermostable polymerase is Taq DNA polymerase. This enzyme is appropriate for routine ampliﬁcations, but the use of other thermostable polymerases can enhance results. Ampliﬁcation reactions also contain buffer, deoxynucleotide triphosphates, and magnesium. The magnesium ion concentration affects enzyme activity, primer annealing, melting temperature of the template and the PCR product, ﬁdelity, and primer-dimer formation (2). How the interaction of these components, cycling parameters, as well as other factors contribute to successful PCR will be discussed in the following chapters.
The Basics of RT-PCR RT-PCR combines cDNA synthesis from RNA templates with PCR (ﬁgure 1) to provide a rapid, sensitive method for analyzing gene expression. RT-PCR is used to detect or quantify the expression of messages, often from small amounts of RNA (3,4). In addition, the technique is used to analyze differential gene expression or clone cDNAs without constructing a cDNA library (5,6). RT-PCR is more sensitive and easier to perform than other RNA analysis techniques, including Northern blots, RNase protection assays, in situ hybridization, and S1 nuclease assays (7,8). The template for RT-PCR can be total RNA or poly(A)+ selected RNA. RT reactions can be primed with random primers, oligo(dT), or a gene-speciﬁc primer (GSP) using a reverse transcriptase. RT-PCR can be carried out either in two-step or one-step
formats. In two-step RT-PCR, each step is performed under optimal conditions. cDNA synthesis is performed ﬁrst in RT buffer and one tenth of the reaction is removed for PCR. In one-step RT-PCR, reverse transcription and PCR take place sequentially in a single tube under conditions optimized for both RT and PCR. This Guide describes the keys to successful RT-PCR and PCR. High sensitivity (getting enough product from small samples) and high speciﬁcity (selective ampliﬁcation of only the desired product) are the hallmarks of successful PCR. Optimal RT-PCR and PCR can be obtained through careful experimental design, including selecting the appropriate enzymes, designing optimal primers, using different buffers and additives, establishing cycling parameters, and preparing high quality templates.
Cells or Tissue
RNA Isolation AAAAAAA(A)n AAAAAAA(A)n AAAAAAA(A)n cDNA Synthesis
gene speciﬁc priming AAAAAAA(A)n AAAAAAA(A)n AAAAAAA(A)n
oligo(dT) priming AAAAAAA(A)n TTTTTTTT AAAAAAA(A)n TTTTTTTT AAAAAAA(A)n TTTTTTTT N6
random hexamer priming N6 N6 N6 N6 N6 AAAAAAA(A)n N6 N6 N6 N6 AAAAAAA(A)n
FIGURE 1. RT-PCR overview.
T H E
B A S I C S
P C R
A N D
R T - P C R
Add RNase inhibitors to the ﬁrststrand reaction in the presence of buffer and a reducing agent. Ampliﬁed product is detectable when either total RNA or poly(A)+ RNA is used as the starting template (ﬁgure 2) (14). such as DTT. In addition. and does not contain inhibitors of reverse transcriptases such as EDTA or SDS (9). simply precipitate it by adding NaCl to 0. dissolve the RNA in deionized formamide and store at –70°C. whereas RNA isolated from rat liver. One popular RNA isolation protocol is the single-step method which uses guanidine isothiocyanate/acidic phenol (10). The ampliﬁed targets were a 377-bp fragment near the 5´ end of the DNA polymerase ε mRNA and a 643-bp fragment of the replication protein A mRNA. RNA from pancreas was stable for at least 1 year in formamide. poly(A)+ RNA may increase the sensitivity of detection when analyzing rare messages. The quality of the RNA dictates the maximum amount of sequence information that can be converted into cDNA. Outline of Procedure for TRIzOL® Reagent RNase inhibitors are often added to RT reactions to help improve cDNA length and yield. B. and C.P C R S E N S I T I V I T Y 3 . High-quality RNA is substantially full length. The formamide used for storage of RNA must not contain impurities that cause RNA degradation (16). transcripts >4 kb are more TRIZOL Reagent can isolate high-quality RNA from as little as 100 cells or 1 mg of tissue. This is especially true for long term storage.C HA AP PT TE ER R 2 2 CH Increasing RT-PCR Sensitivity S Isolating High-Quality RNA uccessful cDNA synthesis starts with high-quality RNA. To increase the stability of stored RNA samples. Oligo(dT) selection for poly(A)+ RNA is typically not necessary. I N C R E A S I N G R T . The TRIZOL® Reagent method (see ﬁgure below) is an improvement on this single-step method and can be used to isolate high quality. In addition. Comparison of total RNA and poly(A)+ RNA in RT-PCR. respectively) with oligo(dT) primers and SUPERSCRIPT™ II RT. but do not protect against RNases found on the skin. which are both moderately abundant targets. due to the carry-over of trace amounts of RNase (15).000 × g for 5 min. even when using these inhibitors be careful not to introduce RNases from your ﬁngertips. 100 bp DNA Ladder Poly(A)+ RNA 3 4 100 bp DNA Ladder – 377 bp – 643 bp Total RNA 1 2 Homogenize sample in TRIZOL Reagent Separate Phases (add chloroform) Panel A Precipitate RNA Panel B Wash and Solubilize Elapsed Time <1 h FIGURE 2. RNA isolated from RNase-rich samples. which can bias the detection or quantiﬁcation of message. respectively) or from 500 ng or 50 ng of poly(A)+ HeLa RNA (lanes 3 and 4. which contains lower amounts of RNases. undegraded RNA from various cells and tissues (11. Thus. since procedures prior to cDNA synthesis that denature the inhibitor will release bound RNases that can degrade RNA. Incubate 3 to 5 min at room temperature and centrifuge at 10. was stable in water for 3 years (15). One tenth of the cDNA reaction was ampliﬁed for 30 cycles using Taq DNA polymerase. isolating poly(A)+ RNA may lead to variability of mRNA enrichment between samples.12). However. The TRIZOL Reagent method can be used to isolate RNA from as little as 100 cells or 1 mg of tissue (13). susceptible to degradation by trace amounts of RNases than shorter transcripts (16). When ready to use the RNA.2 M followed by 4 volumes of ethanol. such as the pancreas. may need to be stored in formamide to maintain high quality RNA. cDNA was synthesized in duplicate from 5 or 1 µg total HeLa RNA (lanes 1 and 2. Protein RNase inhibitors protect RNA against degradation by RNases A. RNA isolated from rat pancreas and stored in water showed substantial degradation after just 1week.
decreasing both the amount and size of the cDNA. SUPERSCRIPT II RT signiﬁcantly increased the yield of long RT-PCR products compared to M-MLV RT (ﬁgure 5) (20). RT-PCR sensitivity can be limited by the amount of cDNA synthesized. RNA template that is cleaved by RNase H activity is no longer an effective substrate for cDNA synthesis. cDNA was synthesized from 2. Effect of RT on sensitivity in long RT-PCR. cDNA was synthesized from total HeLa RNA at 50°C with oligo(dT) primers using THERMOSCRIPT™ RT or AMV RT. Therefore. Figure 3 900 800 cDNA Synthesized (ng) 700 600 500 400 300 200 100 0 AMV RT THERMOSCRIPT RT M-MLV RT SUPERSCRIPT™ II RT. Obstacles Posed by RNase H TTTTTTTT AAAAAAAA TTTTTTTT AAAAAAAA TTTTTTTT SUPERSCRIPT II RT A FIGURE 3. The RNase H activity competes with the polymerase activity for the hybrid formed between the RNA template and the DNA primer or growing cDNA strand and degrades the RNA strand of the RNA:DNA complex (see ﬁgure). an avian RNase H– reverse transcriptase.000 RNA (ng) – 3. yield greater amounts of cDNA and more full-length cDNA than M-MLV RT and AMV RT. especially when cloning large cDNAs. Both moloney murine leukemia virus (M-MLV) and avian myleoblastosis virus (AMV) reverse transcriptases have endogenous RNase H activity in addition to their polymerase activity. PCR was 35 cycles with PLATINUM® Taq DNA Polymerase and primers for human DNA polymerase ε. S M S M kb – 6.5 µg (for THERMOSCRIPT™ RT and AMV) or 1 µg (for SUPERSCRIPT™ II RT and M-MLV) of a 7.C HA AP PT TE ER R 2 2 CH Increasing RT-PCR Sensitivity (continued) Using RNase H– Reverse Transcriptases Reverse transcriptases catalyze the conversion of RNA to cDNA. respectively (ﬁgure 3) (17–19). The size of RT-PCR products is limited by the ability of a reverse transcriptase to synthesize full-length cDNA.000 1 Kb PLUS DNA Ladder™ 10 AMV RT 100 1. TTTTTTTT AAAAAAAA AA AA Effect of RNase H on ﬁrst-strand cDNA. cDNA was synthesized from 5 µg total HeLa RNA with oligo(dT) primers.3 kb) and human DNA polymerase ε mRNA (6. Full-length cDNA ( ) was assayed by cutting and counting size-fractionated bands from alkaline agarose gels.3 FIGURE 5. The synthesis of full-length cDNA for human tuberous sclerosis II mRNA (5.5-kb mRNA using oligo(dT) primer and 10 µCi of [α-32P]dCTP with the recommended synthesis conditions. Effect of RT on yield of ﬁrst-strand cDNA. Samples were treated with RNase H and one tenth of the cDNA reaction was ampliﬁed with ELONGASE® Enzyme Mix for 35 cycles. it is advantageous to eliminate or greatly reduce the RNase H activity of reverse transcriptases. so reactions can be performed above the normal incubation temperature of 37°C to 42°C. 10 THERMOSCRIPT RT 10 1. RNase H degrades RNA in an RNA:DNA complex during cDNA synthesis.8 kb) was catalyzed by SUPERSCRIPT™ II RT (lane S) or M-MLV RT (lane M). THERMOSCRIPT RT has signiﬁcantly greater sensitivity than AMV RT (ﬁgure 4) (18). Total yield of ﬁrst-strand ( ) was calculated from TCA precipitation. Effect of RT on sensitivity of RT-PCR. an M-MLV RNase H– RT. Red arrows represent potential cleavage sites.P C R S E N S I T I V I T Y .8 – 5. RNase H– reverse transcriptases also have increased thermostability. 4 I N C R E A S I N G R T .5 kb FIGURE 4. and THERMOSCRIPT™ RT.
such S – RNase H M A S + RNase H M A – 5. For maximum RT-PCR sensitivity in SUPERSCRIPT II RT reactions. the ﬁnal concentration of glycerol in the ampliﬁcation reaction is 0. RNase H Treatment Treating cDNA reactions with RNase H prior to PCR can improve sensitivity. incubating the RNA and primer at 65°C in the absence of buffers or salts and quickly chilling on ice eliminates most secondary structure and allows primer binding. since planned incubation temperature. TABLE 2. In addition to using nating genomic DNA.3 kb ™ ® FIGURE 7.C HA AP PT TE ER R 2 2 CH elevated RT temperatures. 40 cycles of PCR was performed with one tenth of the reverse transcriptase. reduces sensitivity and. Effect of RNase H treatment on RT-PCR. secondary structure may still exist in some templates even after heat denaturation.5 kb – of Mn++.P C R S E N S I T I V I T Y 5 .3 kb) from 5 µg of total HeLa RNA was catalyzed by SUPERSCRIPT™ II RT (S). which cDNA reaction and PLATINUM Taq DNA Polymerase High Fidelity. cDNA was synthesized at the indicated temperatures from 10 ng of soybean total RNA with THERMOSCRIPT RT or AMV RT and a polymerase is a less efﬁcient gene-speciﬁc primer for 18S rRNA. RNase H treatment is often necessary when amplifying longer. 70°C ﬁrst-stand synthesis temperatures can be used.5% of the starting RNA was ampliﬁed for 35 cycles with ELONGASE® Enzyme Mix. This makes Tth polymerase less suitable for applications that require highly accurate ampliﬁcation. which will not inhibit PCR. which functions as a DNA polymerase in the presence of Mg++ and a reverse transcriptase in the presence 1. If using a GSP. especially when using a gene-speciﬁc primer (GSP) for cDNA synthesis (see page 7). For targets >1 kb. use ﬁrst-strand synthesis temperatures ≤65°C. Do not use a single enzyme is used for both reverse oligo(dT) and random primers above 60°C. RNase H treatment improved the signal from cDNA generated with either SUPERSCRIPT II RT or M-MLV RT. RNase H treatment is optional since incubation at 95°C for the PCR denaturation step often hydrolyzes the RNA in the RNA:cDNA complex. RT incubation temperatures. transcription and PCR. the presence of Mn++ during PCR reduces ﬁdelity. However. AMV RT will also tolerate up to 20% glycerol without loss of activity. Higher temperatures can also improve speciﬁcity. Up to 20% glycerol or up to 10% DMSO can be used without effecting SUPERSCRIPT II RT or M-MLV RT activity (9). Using a thermal cycler can simplify the multiple temperature THERMOSCRIPT RT AMV RT 1 Kb PLUS shifts required for RT-PCR.4%. One half of the reaction was treated with RNase H for 30 min. increase speciﬁcity by shifting the RNA/primer mix directly from the 65°C denaturation temperature to the RT incubation temperature and add prewarmed 2X reaction mix (cDNA synthesis hot start). Additives to Enhance RT Additives including glycerol and DMSO can be added to ﬁrst-strand synthesis reactions to help destabilize nucleic acid duplexes and melt RNA secondary structure. For some targets. add 10% glycerol and incubate at 45°C. The ampliﬁcation of products from these difﬁcult templates can be improved by using THERMOSCRIPT RT and performing the RT reactions at higher temperature (ﬁgure 6) (18). An amount of the treated or untreated RT reaction equivalent to 0. For targets ≤1 kb. DNA Ladder 50°C 55°C 60°C 50°C 55°C 60°C Tth thermostable polymerase. However. In addition. a control reaction random primers require an initial 10 min without RT cannot be used to distinguish incubation at 25°C before increasing the ampliﬁed cDNA from ampliﬁed contamitemperature to 60°C. such as cloning cDNAs. For many RNA templates. can also be incubated up to 65°C. This helps prevent intramolecular base pairing that can occur at lower temperatures. If one tenth of the RT reaction is added to the PCR. full-length cDNA targets. Tth FIGURE 6. The synthesis of full-length cDNA for human tuberous sclerosis II mRNA (5. or AMV RT (A). In these cases. M-MLV RT (M). it is thought that the RNA in the cDNA reaction may prevent binding of the ampliﬁcation primers. Effect of temperature on a difﬁcult template. For many RT-PCR applications. For this difﬁcult target. as the low copy target tuberous sclerosis II (ﬁgure 7) (20). ™ Higher RT Incubation Temperatures Higher RT incubation temperatures (table 2) can help disrupt RNA secondary structure and increase the yield of product (18). I N C R E A S I N G R T . make sure the Tm of the primer is as high as the Table 2 Reverse Transcriptase AMV M-MLV SUPERSCRIPT™ II RT THERMOSCRIPT™ RT Incubation Temperature 37°C – 45°C 37°C 37°C – 50°C 42°C – 65°C* *RNA begins to hydrolyze at temperatures above 65°C. RNase H treatment can improve sensitivity.
Combine with PLATINUM Pfx DNA Polymerase for greater ﬁdelity. 10. Two-Step RT-PCR Two-step RT-PCR is popular and useful for detecting multiple messages from a single RNA sample. Glycogen is water soluble and remains in the aqueous layer with the RNA to aid subsequent precipitation. down to 0. The addition of glycogen as a carrier during RNA isolation helps improve the yield from small samples (13). RNase-free glycogen is added at the same time TRIZOL Reagent is added. TABLE 3. • For one-step RT-PCR.1.1 pg total RNA (ﬁgure 9) (22). Combine with PLATINUM® enzymes for higher speciﬁcity. One-Step vs.5 µg of acetylated BSA using SUPERSCRIPT™ II RT. 30 cycles of PCR was performed with Taq DNA polymerase. then 40 cycles of 94°C for 15 s and 55°C for 30 s and 68°C for 90 s. and helps minimize carry-over contamination since tubes do not need to be opened between cDNA synthesis and ampliﬁcation. Reactions contained 200 nM of sense and antisense primer. 5 fg. Fewer pipetting steps and reduced chances of contamination. and 0. The “no-RT” control contained 50 pg of CAT transcript (lane 7). a one-step RT-PCR method offers other beneﬁts (table 3) (22). one-step RT-PCR can provide greater sensitivity. 5 pg.5 kb. 94°C for 2 min. Table 3 TWO-STEP PROCEDURE Prime ﬁrst-strand cDNA with: Oligo(dT) Random hexamers GSP primers Provides: • Flexibility Choice of primers. • Ideal for detecting or quantifying several messages from a single sample. – 353 bp FIGURE 9. Reactions were incubated at 50°C for 30 min. A β-actin fragment was ampliﬁed from 0.P C R S E N S I T I V I T Y . • Ideal for analysis of large numbers of samples. 500 fg. use an antisense gene-speciﬁc primer (GSP) to prime cDNA synthesis. respectively) of CAT mRNA transcript were reverse transcribed without (Panel A) or with (Panel B) 0. 6 I N C R E A S I N G R T . the addition of BSA or RNase inhibitor to SUPERSCRIPT II RT reactions is still recommended. 50 pg. 102. • Use PLATINUM Pfx DNA Polymerase or PLATINUM Taq DNA Polymerase High Fidelity for products <12 kb. For successful one-step RT-PCR.5 fg (lanes 1 to 6. for small amounts of RNA. 1. respectively) using the SUPERSCRIPT™ One-Step RT-PCR System. Effect of BSA on RT-PCR sensitivity. • Ability to optimize for difﬁcult RT-PCR. • Ideal for quantitative PCR. 1 2 3 4 5 6 100 bp DNA Ladder 500 bp – FIGURE 8. • Use ELONGASE® Enzyme Mix for products >12 kb. ONE-STEP PROCEDURE Prime ﬁrst-strand cDNA with: GSP primers Provides: • Convenience Ampliﬁcation enzymes premixed with reverse transcriptase. Comparison of two-step and one-step RT-PCR. Sensitivity of one-step RT-PCR. and 103 pg of total HeLa RNA (lanes 1 to 6. However. One-step RT-PCR is easier to use when processing large numbers of samples. Choice of ampliﬁcation enzyme. Notes on ampliﬁcation enzymes and product size: • Use Taq DNA Polymerase or PLATINUM Taq DNA Polymerase for products <4 kb. The recommended concen1 2 3 A 4 5 6 7 tration of RNase-free glycogen is 250 µg/ml for <50 mg of tissue or <10 6 cultured cells. 50 fg. The addition of acetylated BSA (figure 8) to RT reactions performed with SUPERSCRIPT II RT can increase sensitivity (21). and use PLATINUM Taq DNA Polymerase High Fidelity for products <9 kb. Since the entire cDNA sample is ampliﬁed. 0. followed by 68°C for 5 min. If glycogen is 1 2 3 B 4 5 6 7 used during the RNA isolation.C HA AP PT TE ER R 2 2 CH Increasing RT-PCR Sensitivity (continued) Improving Detection of Small Amounts of RNA RT-PCR is particularly challenging when only small amounts of RNA are available. Also. reducing the amount of SUPERSCRIPT II RT and adding 40 units of RNASEOUT™ Ribonuclease Inhibitor can enhance the level of detection. use Taq DNA Polymerase or PLATINUM Taq DNA Polymerase for products <3. • High sensitivity.
since secondary structure of the RNA target may prevent primer binding. Higher RT Incubation Temperatures To take full advantage of the speciﬁcity provided by GSPs. if a GSP has an annealing temperature of 55°C. I M P R O V I N G R T . The use of 1 pmole antisense GSP in a 20-µl ﬁrststrand reaction is recommended. instead of annealing to entire RNA populations as with random primers or oligo(dT). However. The relative speciﬁcity of each method inﬂuences the amount and the variety of the cDNA synthesized.3). The starting concentration range is 50 to 250 ng of random primers per 20 µl reaction. Thermostable RTs can be incubated at higher temperatures to increase reaction stringency (17. oligo(dT) priming generally does not require optimization of the primer-to-RNA ratio or poly(A)+ selection. the amount and complexity of cDNA is considerably less than when random primers are used. Oligo(dT)12-18 is suitable for many RT-PCR applications. Because of its higher speciﬁcity. A GSP can be the same sequence as the ampliﬁcation primer which anneals closest to the 3´ end of the message. Since poly(A)+ RNA constitutes approximately 1% to 2% of a total RNA population. which can eliminate the nonspeciﬁc products generated at lower temperatures (ﬁgure 10).P C R S P E C I F I C I T Y 7 .C HA AP PT TE ER R 3 3 CH Improving RT-PCR Speciﬁcity F Priming cDNA Synthesis irst-strand cDNA synthesis reactions may be primed using three different methods. When maximizing speciﬁcity. Minimizing Contaminating Genomic DNA One potential difﬁculty encountered with RT-PCR is genomic DNA contamination of RNA. Products generated in the absence of RT are of genomic origin. A gene-speciﬁc primer (GSP) is the most speciﬁc primer for the RT step. perform a control experiment without RT for each RNA template to determine whether a given fragment is of genomic DNA or cDNA origin. Effect of RT reaction temperature on RT-PCR speciﬁcity. add the RNA/primer mix directly from the 65°C denaturation temperature to the RT incubation temperature and add prewarmed 2X reaction mix (cDNA synthesis hot start). design primers that anneal in separate exons. To maximize the size of the cDNA.18). 50°C 55°C 60°C – 2 kb FIGURE 10. Random primers are the least speciﬁc of the three methods. The same rules for designing PCR primers are applied to the design of the GSP for the RT reaction (see chapter 5). use an RT with greater thermostability. page 5). Some targets require the design of more than one antisense GSP for successful RTPCR. such as TRIZOL Reagent. Oligo(dT) priming is more speciﬁc than random primers. THERMOSCRIPT RT was added to a prewarmed reaction mixture and 35 cycles of PCR was performed using one tenth of the cDNA with PLATINUM® Taq DNA Polymerase. remove the contaminating DNA by treating RNA with DNase I. To terminate the DNase I digestion. cDNA was synthesized from 1 µg of HeLa RNA with THERMOSCRIPT™ RT and a GSP designed to anneal to the human DNA polymerase ε mRNA. To avoid generating products from genomic DNA. For example. This method is often used to capture 5´ end sequences and make cDNA from RNA templates with regions of secondary structure or pause sites that reverse transcriptases cannot copy (2. since it has greater thermostability for use at higher RT incubation temperatures.5 µg of oligo(dT) primer per 20 µl reaction is recommended. In addition. SUPERSCRIPT II RT and THERMOSCRIPT RT allow for reaction temperatures of 50°C and greater (table 2. Using a good RNA isolation technique. minimizes the amount of contaminating genomic DNA in the RNA preparation. partial-length cDNAs. The PCR products derived from cDNA will be shorter than products ampliﬁed from contaminating genomic DNA. This helps prevent intramolecular base pairing that can occur at lower temperatures. incubate the sample at 65°C for 10 min in the presence of 2. or a GSP can be designed to anneal downstream of the reverse ampliﬁcation primer. The primers anneal to multiple sites along the entire transcript to generate short. To distinguish ampliﬁed cDNA from ampliﬁed contaminating genomic DNA. The use of 0. It hybridizes to the poly(A) tails found at the 3´ ends of most eukaryotic mRNAs (23).0 mM EDTA. the speciﬁcity conferred by the GSP is not fully utilized if the RT reaction is performed with AMV RT or M-MLV RT under low stringency at 37°C. The EDTA chelates the magnesium and prevents magnesium dependent hydrolysis of the RNA that can occur at higher temperatures. Using a thermal cycler can simplify the multiple temperature shifts required for RT-PCR. the ratio of primers to RNA may need to be determined empirically for each RNA preparation. Oligo(dT)20 is provided with the THERMOSCRIPT RT-PCR System. Since the majority of the cDNA synthesized from total RNA with random primers is ribosomal. poly(A)+ selected RNA is often used as the template. GSPs are antisense oligonucleotides that hybridize to 1 Kb PLUS DNA LADDER™ speciﬁc RNA target sequences. Ampliﬁcation Grade before reverse transcription.
a Southern blot can be used to detect products.8-kb band. Incomplete cDNA synthesis lowers the yield of full-length product and contributes to the smearing pattern observed with some targets since shorter cDNAs are tailed and ampliAnneal first strand ﬁed.. since only Figure 11 mRNA 5´ GSP1 (A)n 1 2 5´ 3´ (A)n 3´ 5´ 3´CC. or even a smear with no distinguishable products (ﬁgure 12) (28). IG 3´CC. RT-PCR is more sensitive than Northern blot or ribonuclease protection analysis and requires less RNA and sequence information. 1 µl of the tailing reaction was directly ampliﬁed for 40 cycles with primers for human tuberous sclerosis and ELONGASE® Enzyme Mix (primary PCR 2. cDNA was synthesized from 5 µg HeLa total RNA with SUPERSCRIPT™ II RT at 45°C. The gel is stained with SYBR® Green I.P C R A P P L I C A T I O N S . Unlike conventional RT-PCR. 5´ RACE sensitivity is affected by the RT used and secondary structure at the 3´-end of the cDNA that may inhibit cDNA tailing.CC UAP AUAP 5´ 3´ GI .. Summary of the 5´ RACE procedure. 5´ RACE is more challenging and less speciﬁc than RT-PCR applications.. multiple products. which employs two sequencespeciﬁc primers. The quality of the result depends on the speciﬁcity of the GSPs used in ﬁrst-strand synthesis and ampliﬁcation. If no products are visible or if only a smear is visible after nested ampliﬁcation.8 kb. or combined to generate full-length cDNA (25-27). terTM 1 Kb DNA Ladder – kb ––––2.. The use of RNase H– RT and high-ﬁdelity thermostable polymerases allows higher ﬁdelity ampliﬁcations of longer sequences to generate full-length cDNA clones. but the major difﬁculty in quantitative PCR is the exponential nature of PCR. minal deoxynucleotidyl transferase is tolerant of up to 20% DMSO. Nested PCR (30 cycles) was performed using 1 µl of size-selected PCR product (nested PCR 2...7 FIGURE 12. The tailing enzyme. especially for tranDegrade RNA scripts >1 kb (28). but offers advantages over traditional RNA analysis methods.. Doublewith RNase Mix Purify cDNA with stranded 3´ termini and hairpin GLASSMAX Spin Cartridge structures impair tailing of Tail purified cDNA with dCTP and TdT cDNA by decreasing the availability of the 3´-OH for PCR amplify dC-tailed tailing. 5´ RACE. Two popular ® 8 R T . 10 µl of puriﬁed cDNA was tailed in the presence of 10% DMSO. Ampliﬁcation with nested primers (up to three rounds of nested ampliﬁcation) and using size-selected ampliﬁed product as the target in successive rounds of ampliﬁcation increases the speciﬁcity of 5´ RACE (see nested PCR discussion on page 13). RT-PCR involves two enzymatic steps that can contribute to variability in the amount of RT-PCR product.7 kb. used to prepare probes. The amount of RNA converted to cDNA affects yield. IG CC. lane 1). A 10 µl gel plug was removed around the 2. RACE uses one sequencespeciﬁc primer and either the poly(A) tail of mRNAs (3´ RACE) or a homopolymeric tail added to cDNA ends (5´ RACE) (ﬁgure 11). to mRNA generates more full-length cDNA. as well as decreasing the magnesium concentration in the ampliﬁcation reaction. and Some difﬁcult targets may nested GSP require the addition of DMSO for effective tailing (ﬁgure 12) (28). 5´ RACE products may be a single product. directly sequenced. Quantifying mRNA Expression Quantifying mRNA expression is challenging RT-PCR.8 –2. lane 2). RACE has been used for the ampliﬁcation and cloning of rare mRNAs (23-25). The DNA was eluted in 50 µl of TE. it can increase the Copy mRNA into cDNA with level of detection of 5´ end SUPERSCRIPT II RT sequence. Since SUPERSCRIPT II RT primer. However. Increasing the RT incubation temperature and PCR annealing temperature. One method for joining 5´ and 3´ RACE products is to use the sequence information generated by 5´ and 3´ RACE to design new primers which will amplify the entire cDNA sequence (26). The initial incubation cDNA using the Abridged Anchor Primer of the cDNA at 94°C followed and nested GSP2. RACE products can be cloned.. PCR product using AUAP.CC nested GSP GSP2 5´ 3´ 5´ FIGURE 11..C HA AP PT TE ER R 4 4 CH RT-PCR Applications R 5´ and 3´ RACE apid ampliﬁcation of cDNA Ends (RACE) is a procedure to capture unknown sequences at either the 5´ or 3´ end of a transcript. the complexity and abundance of the target. one of the ampliﬁcation primers is genespeciﬁc. the speciﬁcity of the anchor primer during ampliﬁcation. and the length of the product. or UAP. GSP1. can enhance speciﬁcity. by chilling on ice helps to Reamplify primary disrupt secondary structure. This requires internal sequence information to use as a probe.CC 5´ Abridged Anchor Primer 5´ GI . in which small variations between samples translates to large differences in product yield (29)..
The internal standard RNA is reverse transcribed and ampliﬁed with the target to control for differences in the efﬁciency of cDNA conversion and ampliﬁcation.600 the target. The internal standard RNA shares the same primer recognition sites with the target. Fluorescence quenches its signal until the probe is cleaved. Competitive RT-PCR is an end-point analysis that requires the ampliﬁcation efﬁciency of the target and the internal standard to be equal.56 5 Correlation Coefficient: 0. Standard curve. The outer loop structure is messages in a single RNA sample and use a composed of 15 to 30 nucleotides and is one-step strategy for added convenience complementary to the target sequence.581 10 is known as the threshold cycle Y-Intercept: 41.34).993 or CT. a Real-time RT-PCR is non-competitive and detects the product as it is being formed. reactions containing more standard than target and less standard than target are required. Real-time PCR quantitation of retinoblastoma mRNA. When when processing large numbers of samples. This product can be cloned into a vector carrying the T7 or SP6 RNA polymerase promoter. Relative ﬂuorescence intensity of duplicate samples during (ﬁgure 13). The CT value is determined mulation of product including ﬂuorogenic for different concentrations of the in vitro 5´ nuclease probes and Molecular Beacons transcribed RNA. ﬂuorescent 600 400 measurements are made in real Fluorescence Threshold 200 time during each cycle of PCR 0 -200 using a thermal cycler coupled to -400 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 an optical excitation and detection Cycle Number device. The amount of ﬂuoresPanel B cence released is proportional to 50 45 the amount of PCR product. the target sequence is present. These probes are wide range of target concentrations (ﬁgure 13) designed with a hairpin structure bringing (30). relaxing the hairpin 500 ng 50 pg 1. Use a two-step structure is composed of 5 to 7 nucleotides RT-PCR strategy for quantifying multiple and is GC rich. quantitative PCR and realtime PCR (4. Oligo(dT) sequence can be installed by adding poly(dT) sequences to the reverse primer (32). Panel A.accurate with a linear-dose response over a phore and 3´ quencher.P C R A P P L I C A T I O N S 9 .C HA AP PT TE ER R 4 4 CH methods for quantifying mRNA abundance are competitive. The stem target abundance is required. RT-PCR of number samples will have low CT duplicate samples of total HeLa RNA was performed with the PLATINUM Quantitative values and low-copy-number RT-PCR THERMOSCRIPT One-Step System (cDNA synthesis was performed at 60°C) and detected using a FAM-labeled Molecular Beacon probe (400 nM) on an ABI samples will have high CT values Prism 7700. Then the CT values are (33. Several methods exist to generate internal standards (31).30). Real-time RT-PCR offers advantages assay uses probes with two ﬂuorescent labels. but is a different size. standard curve is generated using an in vitro Several probes can be used to assay the accu. For absolute quantiﬁcation of messages. The (ﬁgure 13. The ﬁrst method is based on the 5´ plotted against the RNA concentrations to nuclease activity of Taq DNA polymerase. Thus. an exogenous RNA transcript (internal standard RNA) is added before the RT step to control for sample-tosample variation. the Panel A 2. high-copyFIGURE 13.800 Total HeLa RNA 1. One of the simplest methods involves installing the primer recognition sites on nonspeciﬁc spacer DNA by PCR (31).cation analysis and little prior knowledge of imity to quench ﬂuorescence (34). The abundance of the target is determined by comparing the signal obtained for the internal standard to the signal for the target.transcribed RNA.400 50 ng 5 pg structure and allowing ﬂuores1. Threshold Cycle (CT) Relative Fluorescence 1 2 3 4 5 ® ™ ® 6 R T . Some prior knowledge of the amount of starting target is necessary to establish a concentration range for the internal standard. PCR. The 40 35 cycle at which the amount of 30 released ﬂuorescence crosses an 25 20 established baseline ﬂuorescence 15 Slope: -3.000 cence. one dye serves as a reporter and the other over competitive RT-PCR. quantitative PCR can be performed in one-step RT-PCR using a GSP or performed in a two-step reaction using a GSP or oligo(dT). 800 For both probes. or the RNA promoter sequences can be included on the forward primer in order to install the sequences by PCR (32). panel B). In competitive PCR. For accurate quantitation. Competitive. Then.200 5 ng 0 500 pg 1. generate a standard curve for quantifying the which is used to cleave a hybridization probe level of expression in unknown samples at the branch point during extension (33). Panel B.000 molecular beacon hybridizes to 1. The CT value is inversely 0 proportional to the amount of 10 10 10 10 10 10 Starting Quantity (pg total HeLa RNA) starting target. Quantitation is performed by coampliﬁcation of the speciﬁc target sequence together with known concentrations of the internal standard RNA. monitoring is highly sensitive and highly Molecular Beacons contain a 5´ ﬂuoro. Samples do not require post-ampliﬁthe ﬂuorophore and quencher in close prox. RNA standards can be generated by in vitro transcription.
base is possible. Annealing temperatures are generally set about 5°C below the Tm of the primers. This prevents ampliﬁcation from the primers themselves to form primer-dimers. Longer sequences can hybridize with some mismatching. primers greater than 24 nucleotides do not confer greater speciﬁcity. but high enough to minimize nonspeciﬁc binding. The ﬁrst formula was derived for hybridization in high salt (1 M) and is valid for primers <18 bases. to establish an annealing temperature for Primer pairs whose Tm varies by more than PCR.5 + 16. Poorly designed primers may amplify other. nontarget sequences. This is the ments. check Careful primer design is one of the most important aspects of PCR.C HA AP PT TE ER R 5 5 CH Improving PCR Speciﬁcity C Primer Design areful primer design is one of the most important aspects of PCR. wrong sites. For best results. Higher annealing temperatures can temperature at which 50% of the primer and reduce the formation of both primer-dimer its complementary sequence are present in a and nonspeciﬁc products. Design primers to contain 3 As or Ts within the last 5 nucleotides (35). However. Poorly designed primers may amplify other. Sometimes only limited sequence information is available for primer design. which decreases speciﬁcity. these regions for complementarity and internal secondary structures. The second formula estimates Tm based on GC content and salt concentration. The primer needs to be long enough for the sequence to be unique and to reduce the probability of the sequence being found at non-target sites. Formulas to estimate Tm . Most computer programs degenerate bases at the 3´ end of the primer. In addition. and hybridize slower than shorter sequences. • Avoid sequences with the potential to form internal secondary structure. Tm = 81. However. The Tm can vary signiﬁcantly depending since annealing of the last three bases on the 3´ end can be enough to initiate PCR at the on the formula used and the primer sequence. degenerate primers are designed. can be added to the 5´ end of a primer without affecting speciﬁcity. • Avoid mismatches at the 3´ end. • Avoid complementary sequences at the 3´ end of primer pairs. The following guidelines describe the desirable characteristics of a primer sequence that increase speciﬁcity: • Typical primers are 18 to 24 nucleotides in length. duplex DNA molecule. Ideally. These sequences are not included when estimating the Tm of a primer. the annealing temperature is only a in the degenerate mixture are not speciﬁc for starting point. such as restriction sites and promoter sequences. non-target sequences. Do not include neighboring bases. This increases the primer’s stability and confers hybridization stability with the target sequence. Degenerate primers are a mix of different sequences representing all possible bases coding for a single amino Table 4 acid. • Design primers with G or C residues in the 5´ and central regions. Simple Formula (39) (valid for primers <18 bases) minimize the degeneracy conTm = 4°C × (G + C) + 2°C × (A + T) sulting codon usage tables for base preference in different Tm for Oligonucleotides (40) (dependent on salt concentration) organisms (2). There are several formulas for estimating the Tm (38-40).6 × (log10[Na+]) + 0. if only the amino acid sequence is known. • Select primers that are 40% to 60% GC or mirror the GC content of the template. ingly higher annealing temperatures. Reasonable annealing temperatures range from 55°C to 70°C. To improve speciﬁcity. Additional sequences that are not present on the target. The Tm is necessary both primers need to have a similar Tm. Inosine pairs with all bases and will lower the annealing the primary sequence and the identity of the temperature of the primer. the annealing temperature is 5°C exhibit greater mispriming due to the low enough to guarantee efﬁcient annealing of the primer to the target. Begin at 5°C below the estimated Tm and increase Primer Annealing Temperature Another important parameter for primers is the annealing temperature in 2°C increthe melting temperature (Tm).41 × (%G + C) – 675/n deoxyinosine may be used in Where n = number of bases and [Na+] = monovalent (Na+ or K+) cations places where more than one TABLE 4. Speciﬁcity can be increased by analyzing several reactions with increasthe target (37). The ideal primer pair anneals to unique sequences that ﬂank the target and not to other sequences in the sample. This destabilizes primer annealing. The last 3´ nucleotide needs to anneal to the template for the polymerase to catalyze extension (36). The most reliable method for determining the Tm of a primer is the nearestneighbor analysis (38). This method predicts the hybridization stability of a primer from 10 I M P R O V I N G P C R S P E C I F I C I T Y . Table 4 lists two of the commonly used formulas for determining a primer’s Tm. For example. Use higher primer concentrations Since most formulas provide an estimated (1 µM to 3 µM) because many of the primers Tm value. use nearest-neighbor analysis. • Avoid a GC-rich 3´ end. which may decrease yield (2).
have a greater portion of truncated the primer is essential to accurately deter.5 µM.0). read the optical density at 260 nm (OD260). but only <1 week when (restriction endonuclease sites. the use of any cycle. Life Technologies guarantees cient (OD/µmol) and the reciprocal of the the total oligonucleotide yield as a minimum extinction coefﬁcient (µmol/OD) are pro.14 OD × 100 × 4.9 nmol/OD. Cycling begins with annealing temperature approximately 5°C above the estimated Tm. If two primers have a different Tms. G. These truncated sequences Formula 2 are generated because DNA synthesis chemMillimolar Extinction Coefﬁcient of Oligonucleotide = istry is not 100% efﬁcient. Optimal primer concentration typically falls between 0. Longer primers. Higher concentrations of primer may result in the ampliﬁcation of nonspeciﬁc products. Primer Concentration Primer concentration can affect speciﬁcity. the concentration would be calculated as follows: technology. RNA polymerase promoter sites. desalting is not necExample: To calculate the concentration of an oligonucleotide essary. Primers First-strand cDNA synthesis for RT-PCR Standard reconstituted in TE at ≥10 µM AFLP technology Standard are stable for >6 months at PCR using primers with 5´ sequences Cartridge –20°C.number of OD units (table 6. Primer yield is affected by the efﬁciency mine the concentration (42). It is best to certiﬁcate of analysis. This allows some copies of the target to be generated under more stringent conditions. using Beers Law (formula 1) calculate the concentration by using the absorbance and the reciprocal of the millimolar extinction coefﬁcient (nmol/OD).14 OD and removed by desalting are found in lower the oligonucleotide has a reciprocal extinction coefﬁcient amounts with PARALLEL ARRAY SYNTHESIS of 4. 1 mM EDTA] to bring the ﬁnal bromide-stained gels using oligonucleotides concentration to 100 µM. set the annealing temperature 5°C below the lowest Tm. and T are the number of dAs. Touchdown PCR is useful in applications where the degree of homology between the target sequences and the primer are unknown. and Concentration = 0. such as AFLP® DNA ﬁngerprinting. Failures can occur during extinction coefﬁcient can be used. C. Only targets with the greatest homology will be ampliﬁed.C HA AP PT TE ER R 5 5 CH use of lower annealing temperatures during cycling. using chemical reactions that are repeated for each base added. Minimum recommended primer purity for PCR applications. Then. This is because primers are short and the base composition of the synthesis chemistry and by the method can vary greatly.9 nmol × 1 µmol × 103 ml thus do not interfere with PCR. It is a cyclical A(15. Lyophilized Cycle sequencing Standard Isothermal sequencing Desalted primers are stable >1 year at Site-directed mutagenesis Cartridge –20°C. These products will continue to be ampliﬁed and will compete out nonspeciﬁc products ampliﬁed in later cycles.01) + T(8. To determine primer concentration.4) at pH 8. and dTs (35). The benzoyl and isobutyryl groups in 1 ml. page 12).0 process in which DNA is synthesized 3´→ 5´ Where A. Both the extinction coefﬁ. In addition. Or to increase speciﬁcity.05) + G(12.sequences and may require puriﬁcation. compared to other methods. Touchdown PCR Touchdown PCR increases sensitivity by using stringent annealing conditions during the early cycles of PCR (41). Custom vided on the GIBCO BRL® Custom Primer Primers are shipped lyophilized. etc) stored at room temperature PCR primers >50 bases PAGE (15°C to 30°C). TABLE 5. dGs. and up to 2 months CFLP technology Desalted when stored at room temperature (15°C to 30°C). The stability of the primer Table 5 depends on storage conditions. of the oligonucleotide. especially primers the extinction coefﬁcient calculated for >50 bases. If the A260 = 0. perform 5 cycles at the annealing temperature established by the primer with the higher Tm and then the remaining cycles at the annealing temperature established by lower Tm. TE is better than as standards. Some appliml OD 103 nmol L cations require puriﬁcation to remove any = 69 µM less than full-length sequences generated during synthesis. The millimolar extinction coefﬁcient can be calculated using formula 2 (42). since the staining capability of deionized water since the pH of the water is the standard and the primer can vary greatly often slightly acidic and can cause hydrolysis depending on their sequence (43). do not reconstitute primers in TE [10 mM Tris-HCl estimate primer concentration on ethidium (pH 8. Application Minimum Suggested Purity Store lyophilized and reconstiPCR Standard tuted primers at –20°C. measure the A260 of 10 µl of the oligonucleotide in 990 µl of water (1:100 dilution).1 to 0.2) + C(7. Concentration = A 260 × dilution factor × the reciprocal of the extinction coefﬁcient × conversion factors With Life Technologies® PARALLEL ARRAY SYNTHESIS™ technology.of puriﬁcation. Unlike large doublestranded DNA molecules where an averaged Primer Purity and Stability Standard purity Custom Primers are sufﬁFormula 1 cient for most PCR applications (table 5). ® ™ I M P R O V I N G P C R S P E C I F I C I T Y 11 . It is incrementally decreased by 1°C to 2°C every cycle until the annealing temperature is about 5°C below the Tm. dCs.
Hot start delays DNA synthesis by withholding one of the essential components until the thermal cycler reaches the denatur- Taq DNA polymerase is released into the reaction after incubation at 94°C during the denaturation step.46). Melting of the wax during thermal cycling releases and mixes all of the components together.1 kb of human β-globin from 100 ng of human genomic DNA. such as template and buffer. TABLE 6. Lane 2. FAM. or the construction and manipulation of genetic elements for DNA engineering. Lane 1. but the starting concentration in typical PCR containing 200 µM dNTPs is 1. such as sitedirected mutagenesis. Higher concentrations of free magnesium can result in greater yield. Ampliﬁcation of 4. Taq DNA polymerase with manual hot start by addition of enzyme at 94°C. PLATINUM DNA Polymerases are convenient and highly-effective for automatic hot-start PCR (ﬁgure 14). Improved speciﬁcity with PLATINUM® Taq DNA Polymerase. HEX. Lane 1.5 mM increments. Detection of cloned HIV DNA in human genomic DNA. The antibodies prevent enzyme activity during PCR set-up and even during prolonged incubations at room temperature. wax barrier methods can be cumbersome. The dNTPs and template bind magnesium and reduce the amount of free magnesium available for enzyme activity. PAGE puriﬁcation is recommended and HPLC is not an option. Panel B. and primer annealing. A popular method that limits Taq DNA polymerase activity is to set-up ampliﬁcation reactions on ice and place them into a preheated thermal cycler. Most manual hot-start methods involve the delayed addition of Taq DNA polymerase. and phosphorothioates (S-Oligos). Taq DNA polymerase with room temperature assembly. expression cloning. *These yields are seen with the following 5´ modiﬁcations: biotin. in addition to good primer design. 4. Lane 2. Greater than 90% of Taq DNA polymerase activity is restored in 2 min at 94°C with PLATINUM Taq DNA Polymerase (48). which can affect speciﬁcity. such as magnesium or enzyme. This method is simple and inexpensive. Other modiﬁcations may have slightly lower yields. In comparison to chemically modiﬁed Taq DNA polymerase for hot-start PCR.5 mM (Note: for real-time quantitative PCR use 3 to 5 mM magnesium with ﬂuorescent probes) (2). which can affect yield. PLATINUM Taq DNA Polymerase with room temperature assembly. the polymerase has activity at room temperature (44). Even though the optimal extension temperature for Taq DNA polymerase is approximately 72°C. Taq DNA polymerase with assembly on ice and placed in a preheated (80°C) thermal cycler. primary amines (NH2). Minimum oligonucleotide yield for the various puriﬁcation methods. from each other. which can be cumbersome especially for high-throughput applications (46). for increasing PCR speciﬁcity. longed incubation at 94°C (10 to 15 min) to activate the polymerase. perform a magnesium titration using from 1 mM to 3 mM in 0. but does not completely 1 2 3 1 2 3 ation temperature. PLATINUM Taq DNA Polymerase with room temperature assembly. Like manual hot-start methods.C HA AP PT TE ER R 5 5 CH Improving PCR Speciﬁcity (continued) 100 bp DNA Ladder Hot Start Hot-start PCR is one of the most important methods. Thus. 1. ﬂuorescein (FITC). Magnesium Concentration Magnesium affects several aspects of PCR including DNA polymerase activity.50). Lane 3. To determine the best concentration. rhodamine. Taq DNA polymerase with room temperature assembly. Other hot-start methods use wax barriers to encapsulate an essential component.000 copies of plasmid DNA with the HIV gag region was mixed with 100 ng of genomic DNA and ampliﬁed with primers to the gag region. but can also increase nonspeciﬁc ampliﬁcation and reduce ﬁdelity (49. NA is not available. PLATINUM enzymes do not require pro- 12 I M P R O V I N G P C R S P E C I F I C I T Y 1 Kb DNA Ladder . restoring full polymerase activity. PLATINUM Taq DNA Polymerase is comprised of recombinant Taq DNA polymerase complexed with monoclonal antibodies to Taq DNA polymerase (47). To reduce the Table 6 Minimum Yield (OD) for Different Primer Purities* Number of Bases <20 Synthesis Scale 50 nmole 200 nmole 1 µmole 10 µmole 50 nmole 200 nmole 1 µmole 10 µmole Standard/ Desalted 2 8 20 200 5 20 50 500 Cartridge 2 8 20 NA 2 10 25 NA HPLC NA 3 10 100 NA 3 15 150 PAGE NA 1 3 30 NA 1 5 50 ≥20 Note: For primers >50 bases. The optimal magnesium concentration varies for each primer pair and template. or to physically separate reaction components. Lane 3. TET.1 kb – –114 bp Panel A Panel B FIGURE 14. inactivate enzyme activity and therefore does not completely eliminate the ampliﬁcation of nonspeciﬁc products. Panel A. nonspeciﬁc products are often generated during PCR set-up and at the start of thermal cycling when reactions are brieﬂy incubated at temperatures well below the annealing temperature (45. Once these nonspeciﬁc products are formed they can be efﬁciently ampliﬁed. phosphate (PO4). Hot-start PCR is particularly effective when the sites available for designing primers are limited due to the location of genetic elements. prone to contamination and unreliable in high-throughput applications.
100 bp DNA Ladder 0X 1X 2X 2. some targets. the initial ampliﬁcation product can 2.8 kb – be size selected by gel puriﬁcation.2 2. 60°C for 30 s. 2. Titration of PCRx Enhancer Solution.6 3. require additional measures.0 1. 1.000 and added to the second round of ampliﬁca(mM MgCl2) (mM MgCl2) tion with 15 to 20 1. and increase the speciﬁcity of of secondary structure (53). secondary structure can prevent multiple targets is reduced with nested primer binding and enzymatic elongation. whereas DMSO.8 2. Their proposed mechanism is often ampliﬁes nonspeciﬁc targets. glycerol. use PLATINUM Taq DNA Polymerase.5X 3X 3. formamide.4 1. and magnesium concentrations is adequate to achieve high speciﬁcity ampliﬁcation of many targets.2 2. was ampliﬁed from 100 ng of human genomic DNA using PLATINUM® Taq DNA Polymerase. and PCRX performing the same total number of cycles Enhancer Solution can enhance ampliﬁ.C HA AP PT TE ER R 5 5 CH 1 2 3 4 1 2 3 4 need for magnesium optimization. primer design.0. However. Panel B.0 1. PLATINUM Taq DNA Polythat bind to the target merase with room temperature assembly.5. I M P R O V I N G P C R S P E C I F I C I T Y 13 . Panel A.5X 4X – 149 bp 78% GC FIGURE 17. Improved speciﬁcity with PCRx Enhancer Solution. –156 bp 62% GC Nested PCR Sequential rounds of ampliﬁcation using nested primers can improve speciﬁcity and sensitivity (3). The ﬁrst round is a standard ampliﬁcation of 15 to 20 cycles. Cycling was 35 cycles of 94°C for 30 s. A 149-bp (78% GC) trinucleotide repeat-containing sequence was ampliﬁed from 100 ng of human genomic DNA with PLATINUM® Taq DNA Polymerase in 1X PCRx Ampliﬁcation Buffer with varying amounts of PCRx Enhancer Solution (0 to 4X). A 156-bp fragment (62% GC content). Thus. combining PLATINUM technology and additives enhances speciﬁcity while minimizing the need for the third approach-magnesium optimization. PLATINUM Taq DNA Polymerase functions over a broader range of magnesium concentration than Taq DNA polymerase so less optimization is required (ﬁgure 15) (47). such as amplifying a rare DNA polymerase extend through regions message. and glycerol that inhibit Taq DNA polymerase (46. just inside the ﬁrst required to obtain the best results. A small aliquot of the initial ampliﬁcation is diluted 1:100 to 1:1. betaine.Taq DNA polymerase with assembly on ice and placed in a preheated (80°C) thermal cycler.4 1. Addi. The second Panel B Panel A ampliﬁcation uses a FIGURE 15. 2. and 68°C for 1 min. Two different primer sets are used for the two rounds of ampliﬁcation. optimize the concentration of the additives (ﬁgure 17).8 2.0 1.52). Additives that affect DNA melting temperature provide another method for improving product speciﬁcity and yield (ﬁgure 16). Broader magnesium range with PLATINUM Taq DNA Polymerase.5 mM. Alternatively. thereby PCR can increase the sensitivity from limited aiding primer annealing and helping the amounts of target. especially DMSO.set of primers. It requires less as 5´ RACE.(30 to 40) with the same set of primers cation (51-53). MgCl2 was titrated (1. Complete denaturation of the template is magnesium optimization and works with PLATINUM Taq DNA Polymerase (ﬁgure 16) and PLATINUM Pfx DNA Polymerase.0 cycles. Nested to lower the melting temperature. lanes 1 to 4 respectively) using: standard PCR buffer (panel A) or PCRx Ampliﬁcation Buffer with 1X PCRx Enhancer Solution (panel B). The chance of amplifying tionally.8-kb region of the nested set of primers human β-globin gene was ampliﬁed from 100 ng of human genomic DNA. mentary to both sets of primers. For best results. including formamide.6 3. PCR since fewer targets will be complePCR additives. including those with a highGC content. PCRX Solution more challenging PCR applications such has additional beneﬁts. Additives to Enhance PCR Optimization of the annealing temperature. A 2. ® Panel A Panel B FIGURE 16.0.
which is much smaller. can inhibit ampliﬁcation reactions at concentrations as low as 0.001 0.5 1. 8. and the FTA® Products. Methods for genomic DNA isolation. Reagents such as SDS.01%.6 × 105 3.5 × 107 1.4 kb in lanes 1 to 5.0 × 107 7. The optimal amount of template required depends on the size of the genome (table 8) (56). coli Saccharomyces cerevisiae Arabidopsis thaliana Drosophila melanogaster Homo sapiens Xenopus laevis Mus musculus Zea mays pUC 18 plasmid DNA * Haploid genome size TABLE 8.9 × 109 3. FIGURE 18. 100 ng to 1 µg of human genomic DNA.0 1. DNA was ampliﬁed directly from 1 to 3 mm punches of washed FTA Cards in 50 µl using Taq DNA polymerase (panel A).3 × 109 1. For high-sensitivity PCR of long products (up to 12 kb). which are used in standard genomic DNA preparations.1 Taq DNA Polymerase PLATINUM Taq DNA Polymerase PLATINUM Taq DNA Polymerase High Fidelity Template Concentration The amount of starting template is important for obtaining good product yields.0 × 104 3.0. Enzyme Choice In addition to using high-quality template DNA. which is a matrix-bound method for the storage and puriﬁcation of DNA from blood and other biological samples (table 7) (55). the choice of polymerase also affects yield (see table on page 22).7 × 105 6.3 × 107 6. Table 7 Method Proteinase K/phenol extraction DNAZOL® Reagents FTA® Products TABLE 7. This enzyme combines the beneﬁts of PLATINUM technology with those of enzyme mixes (Taq DNA Polymerase mixed with a proofreading polymerase). For most ampliﬁcations. 10 4 to 10 6 starting target molecules allows sufﬁcient ampliﬁcation to visualize the product on an ethidium bromide-stained gel (2). correlating to 3 × 10 4 to 3 × 105 molecules.2 × 105 3.0 2. such as PLATINUM Taq DNA Polymerase High Fidelity.0 × 107 1.5.69 × 103 Target Molecules/µg of Genomic DNA 1. Human blood was spotted on FTA® Cards. choose an enzyme mix. phenol-free DNA isolation Matrix-bound puriﬁcation and storage Table 8 Genomic DNA E. For plasmid DNA. 7. which are guanidine-detergent lysing solutions. and 8.01 0.01 0.8 × 108 4. PLATINUM polymerases provide better yield than other polymerases because they prevent ampliﬁcation of nonspeciﬁc product during PCR set-up (ﬁgure 18). PLATINUM® Taq DNA Polymerase (panel B). Description Classic method Fast.6 × 108 2.1.8 × 109 2. is sufﬁcient to detect a PCR product from a single-copy gene. Size (bp)* 4.0 1 × 10 – 6 14 I N C R E A S I N G P C R S E N S I T I V I T Y .C HA AP PT TE ER R 6 6 CH Increasing PCR Sensitivity T Template Quality emplate quality affects product yield. respectively. Ampliﬁcation with different thermostable polymerases. A number of contaminants found in DNA preparations can inhibit PCR (46).2. and PLATINUM Taq DNA Polymerase High Fidelity (panel C).7 × 10 6 2. 1 Kb PLUS DNA LADDER™ 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 – kb – 8.1 × 105 2. preferably in a PLATINUM format.5 × 1010 2.4 × 1011 Amount of DNA (µg) for ~10 5 Molecules 0.4 – 4. the amount of DNA added to PCR is in the picogram range. Ampliﬁed products were 4. 5.0 1. Newer methods for isolation of high-quality genomic DNA include DNAZOL® Reagents (53). Correlation of genome size and number of molecules. For example.
1 unit of Taq DNA Polymerase with set-up on ice. Panel A. lanes 1 to 6) was ampliﬁed with primers for a 1. I M P R O V I N G F I D E L I T Y 15 . However. High sensitivity and speciﬁcity with PLATINUM® Pfx DNA Polymerase. Panel C. Panel B. and 0 ng. Decreasing the concentration of dNTPs from 200 µM to 25-50 µM can increase accuracy. high dNTP or magnesium concentrations can reduce ﬁdelity. Other Parameters Besides enzyme.C HA AP PT TE ER R 7 7 CH Improving Fidelity 1 Kb PLUS DNA LADDER™ 1 Kb PLUS DNA LADDER P Enzymes with Proofreading CR applications involving cloning and sequence analysis. these polymerases can give lower yields than Taq DNA polymerase. since the probability of a mutation increases with increasing cycle number and product length. 5. or site-directed mutagenesis require high-ﬁdelity PCR. 1 unit of PLATINUM Pfx DNA Polymerase with room temperature set-up. 50. the ﬁdelity will be effected. PLATINUM Taq DNA Polymerase High Fidelity has 6-times greater ﬁdelity than Taq DNA polymerase alone and can amplify up to 12 kb. The GIBCO BRL high ﬁdelity enzyme mix. If the concentration is not the same for all four nucleotides. Taq DNA polymerase is considered a lowﬁdelity polymerase since it lacks 3´ to 5´ exonuclease (proofreading) activity. PLATINUM Pfx DNA Polymerase has signiﬁcantly better ﬁdelity than Taq DNA polymerase and offers the advantage of high yield and speciﬁcity of PLATINUM products (ﬁgure 19) (59). 10. The use of thermostable polymerases with 3´ exonuclease activity improve ﬁdelity (57. Performing fewer cycles of PCR also can help increase ﬁdelity.1 kb Panel A Panel B Panel C FIGURE 19.1-kb fragment of human thrombospondin for 35 cycles. 2. expression of PCR products. Genomic DNA (100. 1 2 3 4 5 6 1 2 3 4 5 6 1 Kb PLUS DNA LADDER 1 2 3 4 5 6 – 1. Enzyme Mixes Mixing Taq DNA polymerase with a second polymerase with 3´ exonuclease activity provides greater ﬁdelity than Taq DNA polymerase alone and allows for higher yield and ampliﬁcation of longer templates.5 units of PfuTurbo™ DNA Polymerase with set-up on ice.58). 1.
Use aerosol-resistant tips to prevent aerosols from reaching the pipette barrel. a silicabased technology for rapid isolation of DNA fragments from gels. with primers 18 to 24 bases giving good product yields (60. without template. This enzyme (also known as uracil-N-glycosylase or UNG) removes uracil residues from DNA. Completed PCRs containing ~1 µg of ampliﬁed product were spiked with 1. which decreases the yield of longer products. from 80 bp to 10 kb. PLATINUM Pfx DNA Polymerase (has 3´→5´ exonuclease activity) can also amplify longer products (≤12 kb).2 µg of primer (lane 1) or 3. and thermostable polymerases can inhibit cloning. However. the DNA can depurinate. One method to prevent carry-over contamination uses uracil DNA glycosylase (UDG) (62). When cloning PCR products by restriction endonucleases. presumably because it lacks 3´→ 5´exonuclease activity and cannot correct dNTP misincorporations (60). If 3´-recessed termini are generated by digestion. Thus.5 µg of primer (lane 2). minimize the denaturation time at 94°C to 30 s or less during each cycle and limit the preampliﬁcation denaturation time to 94°C for ≤1 min.61). and buffer pH of the standard protocol. Since the elongation times are long. TAQUENCH™ PCR Cloning Enhancer provides an alternative to the clean-up of PCR products. This is called carry-over contamination. and aliquots of the eluate before (lane A) and after (lane B) puriﬁcation were analyzed by agarose electrophoresis. Mixing Taq DNA polymerase with a thermostable DNA polymerase containing 3´→5´ exonuclease activity can allow The Concert Rapid PCR Puriﬁcation System puriﬁes PCR fragments from 80 bp to 10 kb. the ampliﬁcation of longer products requires changes to the extension times. a buffer with a higher initial pH is used. Use premixed reaction components instead of adding each reagent to individual reactions. If the pH falls below pH 7. Spiked reactions were puriﬁed. and labeling reactions. A common source of contamination occurs when previously ampliﬁed products are introduced into new ampliﬁcation reactions. by gel electrophoresis. Substituting deoxyuracil for thymine in ampliﬁcation reactions allows previously ampliﬁed products to be distinguished from template DNA. Puriﬁcation of PCR Products Residual reaction components including primers. Primers were 36 to 40 bases. Taq DNA polymerase does not efﬁciently amplify longer targets (>5 kb). Increase extension times to 1 min/kb to allow the polymerase to complete synthesis. up to 20 min for a 20-kb target. Carry-over contamination can be minimized by using good laboratory procedures during PCR. use an enzyme or enzyme mix for long PCR to obtain the best yield (see table on page 22 and ﬁgure 18 on page 14). The desired product is puriﬁed from the agarose using the CONCERT Gel Extraction System. Puriﬁed DNA from other samples and cloned DNA can also be sources of contamination. from nonspeciﬁc PCR products that can interfere with cloning or sequencing. The elongation rate from a mismatch is greatly reduced. it is often necessary to purify PCR products before further manipulation. residual Taq DNA polymerase and dNTPs from the PCR can ﬁll in the 3´-recessed termini (65).61). Always perform a negative control. ampliﬁcation of targets up to 30 kb (61). Some PCR products may require puriﬁcation. This lowers the cloning efﬁciency and generates clones ligated in the improper reading frame. The CONCERT™ Rapid PCR Puriﬁcation System puriﬁes PCR products from reaction components in 10 min. Clean-up processes involving multiple phenol:chloroform extractions followed by PEG precipitation (63) or isopropanol pre- cipitation (2) are effective but time consuming and can result in product losses. To protect the template against damage. and provides efﬁcient recovery of up to 95% of the original sample (ﬁgure 20) (64). to check for contamination.7 – primers FIGURE 20. In addition to choosing the correct thermostable polymerase. Small amounts of contaminating DNA from an exogenous source can be ampliﬁed along with the desired template. Since the previously ampliﬁed products are susceptible to UDG. High DNA MASS™ Ladder 1 A B A 2 B – kb –3 – 0. It is effective for a wide range of PCR fragments. Prevention of Carry-Over Contamination PCR is susceptible to contamination problems because it is a sensitive ampliﬁcation technique. Primers are designed in the same manner as those used in a standard protocol. Typically the extension temperature is lowered to 68°C for more effective long PCR (60. the addition of TAQUENCH Enhancer before restriction digestion minimizes Taq DNA polymerase activity for efﬁcient cloning (65). sequencing. nucleotides. Designate separate areas for PCR sample set-up and post-ampliﬁcation analysis and change gloves before preparing new reactions. denaturation times.0. 16 O T H E R T H I N G S T O C O N S I D E R .C HA AP PT TE ER R 8 8 CH Other Things to Consider W Amplifying Long Targets hen amplifying targets >5 kb. Puriﬁcation of PCR fragments following ampliﬁcation. newly assembled reactions are treated with UDG before PCR to destroy carry-over products.
Using the indicated primer and template concentration. However. The frac. tion of products in an ampliﬁcation reaction The template quality can also affect containing an extra nucleotide is sequence AFLP results. Multiplex ampliﬁcation of the dystrophin gene was performed with 5 different primer DNA ﬁngerprints generated sets from human genomic DNA with Taq DNA polymerase (panel A) or PLATINUM Taq DNA Polymerase (panel B).nucleotides to amplify an appropriate otide repeat region can be challenging since number of DNAs. Ecotypes were Holladay. Poor quality template may and primer dependent and can vary greatly. Figure 23 5´ 3´ GAATTC CTTAAG TTAA AATT +Eco R I Mse I AATTC G TTAA Eco R I adapter T AAT +Eco R I adapter Mse I adapter TA 3´ 5´ ® Mse I adapter primer +1 5´ A AATTCN TTAAGN NTTA NAAT C 5´ preselective amplification with Eco R I primer +A Mse I primer +C primer +3 5´ AAC AATTCA TTAAGT GTTA CAAT AAC 5´ selective amplification with primers +3 AATTCAAC TTAAGTTG TTGTTA AACAAT denaturing polyacrylamide gel electrophoresis Mse I adapter sequences Eco R I adapter sequences FIGURE 23. PCR-based methods. AP-PCR and RAPD are faster. PLATINUM Taq DNA Polymerase provides greater sensitivity for multiplex and informative method.000 – 500 – 0– Taq DNA Polymerase (66% n + 1) 155 160 n+1 n PLATINUM GENOTYPE Tsp DNA Polymerase (0% n + 1) n n+1 FIGURE 22. resulting However. Schematic of the AFLP® Analysis System.000 – 1. Ampliﬁcation of nga106 locus of Arabidopsis thaliana generated with Taq DNA polymerase (66% n + 1) or PLATINUM® GENOTYPE Tsp DNA Polymerase (0% n + 1). The polymorphism of soybean ecotypes was performed using the AFLP System I and the selective primers Eco R I + ACC and Mse I + CAA. including the number of selective nucleotides in the primers (69).AFLP technique is tolerant of variation in racy of allele size determination (ﬁgure 22) template concentration. Morgan. PI83495. with no difference (66).500 – 1. P C R A P P L I C A T I O N S 17 . A number of parameters inﬂuence AFLP Polymerase over a broad range of magnesium concentrations increases success in results. inhibit restriction endonucleases. PCR.fragments are generated from digesting a plated nucleotide to PCR products. Bass.000 – 500 – 0– 2. RFLP. P83. A typical AFLP® fingerprint. since more restriction Taq DNA polymerase often adds a nontem. of ampliﬁed bands decreases when the Genotyping with number of selective nucleotides increases.500 – 1. but both are sensitive to reaction conditions 547– 459 —— 416 – which can affect the repro360 —— ducibility of the techniques.500 – 2. the nucleotide addition to help increase the accu.C HA AP PT TE ER R 9 CH 9 PCR Applications Tools for Detecting Polymorphisms Ampliﬁed Restriction Fragment Polymorphism (AFLP) technology is a technique for ﬁngerprinting genomic DNA from plants and microorganisms (ﬁgure 23) (68.69). and RAPD are also common DNA ﬁngerprinting techniques. Comparison of DNA polymerases in genotyping of dinucleotide repeat markers. AP-PCR. The speciﬁc DNA sample or to assess the simiincreased robustness of PLATINUM Taq DNA larity between samples (ﬁgure 24) (69). 268 – AFLP technology combines the reliability of RFLP and the convenience of a PCR-based ﬁngerprinting Panel B Panel A method for a more robust FIGURE 21. PLATINUM GENOTYPE Tsp DNA in incomplete digestion and fewer ampliﬁed Polymerase exhibits minimal nontemplated bands visible on the gel (69). Essex. T135.000 – 1. 1 2 3 4 5 6 7 8 9 FIGURE 24. PI90763. be used as a tool to identify a if reactions are not optimized correctly. RFLP is a hybridization-based method primer (nM) 250 250 200 200 100 200 200 100 template (ng) 100 100 250 500 500 250 500 500 which is time consuming and M M bp requires a large amount of genomic DNA. A false negative result may be obtained Figure 21 Nucleotides 150 2. The number multiplex reactions (ﬁgure 21). Dinucleotide Repeat Markers Larger genomes require more selective Determining the size of the ampliﬁed dinucle.larger genome.500 – 2. products with AFLP technology can ranging from 268 bp to 547 bp were ampliﬁed from human genomic DNA.100 ng to 5 µg. Hytest. P88. lanes 1 to 9. multiple primer sets are used simultaneously to amplify several different loci. PI88788. P90. merase in these ampliﬁcations. I Fluorescence Intensity Multiplex PCR n multiplex PCR. respectively. Simply substitute PLATINUM GENOTYPE observed with amounts of DNA between Tsp DNA Polymerase for Taq DNA poly. This complex PCR often results in low product yield and requires higher concentrations of magnesium (67).
25 µg to 0.5 µg acetylated BSA in ﬁrst-strand cDNA synthesis (21). RNase H treat ﬁrst-strand cDNA before PCR (14). Design primers with similar Tms. Possible Cause RNA was degraded Suggested Solution Analyze RNA on a denaturing gel before use to verify integrity. and formamide can inhibit Taq DNA polymerase. Store RNA in 100% formamide (15). Poor PCR primer design DNA contains inhibitors GC-rich template Template concentration is too low Magnesium concentration is too low 18 T R O U B L E S H O O T I N G G U I D E . Use good aseptic technique for RNA isolation. Reagents such as DMSO. For <50 ng RNA. Determine the optimal magnesium concentration for each template and primer pair by performing a reaction series from 1 mM to 3 mM in 0. Glycogen (0. Note: Do not use M-MLV RT above 37°C.1 µg to 0. Note: Do not use oligo(dT) as a primer over 60°C and choose a GSP that will anneal at your reaction temperature. Inhibitors of RT include: SDS. sodium pyrophosphate.0 or inhibitor may release any bound RNases. and guanidinium salts (9). try another GSP or switch to oligo(dT) or random hexamers.C HA AP PT TE ER R 1 10 0 CH Troubleshooting Guide Problem RT-PCR Sensitivity: Little or no RT-PCR product visible after agarose gel analysis. Increase the amount of RNA. Try a different target or tissue. keep reaction temperature ≤ 65°C. Note: Use 3 mM to 5 mM magnesium for real-time quantitative PCR. For gene-speciﬁc primers (GSP). a 10 min incubation at 25°C is recommended before incubating at reaction temperature. glycerol. use 0.4 µg/µl) can be included to aid in RNA recovery for small samples. Also. RNA contained an inhibitor of RT Polysaccharide coprecipitation of RNA Primer used for ﬁrst-strand cDNA synthesis did not anneal well Not enough starting RNA RNA template had high secondary structure The primers or template are sensitive to remaining RNA template Target not expressed in tissue analyzed PCR did not work PCR Sensitivity: Little or no PCR product visible after agarose gel analysis. Remove inhibitor by ethanol precipitation of the RNA. Avoid complementary sequences at the 3´ end of primers. SDS. For random hexamers. Test for inhibitors by mixing a control RNA with the sample and comparing yields to control RNA reaction. do not heat >45°C or use >pH 8. Raise the RT reaction temperature up to 50°C for SUPERSCRIPT II RT or up to 65°C for THERMOSCRIPT RT. spermidine. formamide. EDTA. If inhibitor contamination is suspected. Process tissue immediately after removal from animal. Start with 104 copies of the target sequence to obtain a signal in 25 to 30 cycles. Avoid sequences that can form internal hairpin structures. Denature/anneal RNA and primers in the absence of salts and buffer. For two-step RT-PCR. ethanol precipitate the DNA sample. If using placental RNase inhibitor. Make sure GSP is the antisense sequence. For templates >50% GC content.5 mM increments. Precipitate RNA with lithium chloride to remove polysaccharides. For RT-PCR products >1 kb. use PCRx Enhancer Solution. DTT must be present when the RNase inhibitor is added at ≥ 0. Be sure annealing temperature is appropriate for your primer. Include a 70% (v/v) ethanol wash of the RNA pellet. Use random hexamers in the ﬁrst-strand reaction if full-length cDNA is not needed. do not use more than 1/5 of the RT reaction in the PCR step.8 mM DTT.
C HA AP PT TE ER R 1 10 0 CH Problem PCR Sensitivity: Little or no PCR product visible after agarose gel analysis. Prepare a new deoxynucleotide mix and ensure that the concentration of all four nucleotides is equal or use a prepared mix. Use aerosol-resistant tips and UDG (see page 16). followed by lower annealing temperatures. Use nested primers or touchdown PCR. Use higher annealing temperatures for the ﬁrst few cycles. Contaminating DNA from an exogenous source Primer binding sites are inaccessible due to secondary structure PCR Fidelity: PCR induced errors found in the product sequence Polymerase has low ﬁdelity Too many cycles The concentration of all four deoxynucleotides is not equal Optimize magnesium concentration for each template and primer combination. Check for DNA contamination with a control reaction without RT. For templates >50% GC content use (1X-3X) PCRX Enhancer Solution. Primer concentration is too low RT-PCR Speciﬁcity: Unexpected bands after gel analysis Nonspeciﬁc annealing of primers to templates Poor GSP design Genomic DNA contamination of RNA PCR Speciﬁcity: Unexpected bands after agarose gel analysis Primer-dimer formation Nonspeciﬁc annealing of primers to template Magnesium concentration is too high Primer mispriming due to ampliﬁcation from complex templates. Use a proofreading thermostable polymerase such as PLATINUM Pfx DNA Polymerase. T R O U B L E S H O O T I N G G U I D E 19 . Avoid 2 or 3 dGs or dCs at the 3´ end of primers.5 µM. calculate the concentration using the absorbance and the extinction coefﬁcient (see page 11). Since these equations estimate Tm values. Ampliﬁcation Grade (see page 7). Design primers without complementary sequences at the 3´ ends. (continued) Possible Cause Annealing temperature is too high Suggested Solution Use the equations listed in table 4 (page 10) to estimate the Tm and set the annealing temperature 5°C below the Tm. Use PLATINUM Taq DNA Polymerase for automatic hot-start PCR (47). Try a GSP that allows cDNA synthesis at high temperatures. Optimal primer concentration is between 0. Then. Treat RNA with DNase I. Follow the same rules as described for ampliﬁcation primers. Increase the annealing temperature in 2°C to 5°C increments and minimize the annealing time. To accurately determine primer concentration. Use a GSP instead of random hexamers or oligo(dT) for ﬁrst-strand synthesis. the true annealing temperature may actually be higher or lower. Reduce cycle number. read the optical density at 260 nm (OD260).1 µM to 0.
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L O C A T I N G F O C U S R E F E R E N C E S 21 . just double click on the ﬁle on your local drive and follow the prompts. click on it. you will need the latest Adobe Acrobat Reader. Cumulative FOCUS Subject Index There are 2 ways to ﬁnd articles on a speciﬁc technique: 1. you can download it for free at: www. Instructions to Authors Looking for information on submitting articles to FOCUS ? Check out the Instructions to Authors ﬁle in the FOCUS section of our web site. Viewing a FOCUS Article from our Web Site Finding the Article: When you click on the FOCUS journal on the Home Page. Now you can locate the FOCUS article. Just use the Search box at the top of the FOCUS section of the web.com.) 2.com/products/acrobat/readstep.lifetech. Follow Adobe’s directions for downloading the proper ﬁle to your local drive. If you do not currently have the Acrobat Reader. There are 2 stages to the process: 1.html There is a link to this site in the paragraph at the top of the FOCUS table of contents. (Older issues not displayed there are found by clicking Archives). Follow the procedure to download the Acrobat Reader. These articles contain many details related to protocols to obtain data presented in this Guide. can be obtained from Literature Fulﬁllment through your local Life Technologies Ofﬁce. Note: To install the reader. You can ﬁnd this document on our web site or contact Literature Fulﬁllment through your local Life Technologies Ofﬁce. There is also a search box in the upper left which will allow you to do a full text search of FOCUS articles. and it will open on your screen.C HA AP PT TE ER R 1 11 1 CH Locating FOCUS References Many of the References listed on the previous page are FOCUS® articles. the table of contents for the most recent issue will appear with old issues in the left box. The reader is accessible to you for future use. unless you remove the program from your computer. Locate ﬁle you saved and install the reader on your computer. A Cumulative Subject Index is available for volumes 5-21. 2. You will not need to download the reader again. Viewing the Article: To view the article. (Note ﬁle location when saving.adobe. Locating Previous FOCUS Articles FOCUS Volumes 16— Current Issue can be found on our web site at www. Several of the Classic FOCUS articles (most requested by you through Technical Services) can also be found on the web. Other articles. The web site has a built-in subject index for the articles on the web.
14. PCRX Enhancer System.14 (Consists of PLATINUM Taq DNA Polymerase.15 PLATINUM Taq Antibody *.12 (Consists of Taq DNA Polymerase. PCRX Enhancer Solution. PCRX Enhancer System.184.108.40.206 PLATINUM Pfx DNA Polymerase*.2. 10X Pfx Ampliﬁcation Buffer. 10X PCR Buffer. and MgCl2) PLATINUM Taq PCRX DNA Polymerase*. 10X PCR Buffer. Size <5 kb <5 kb <5 kb <5 kb <5 kb <5 kb <5 kb <12 kb <12 kb <12 kb <0.2. No.2.12. ideal for high-GC content or other problematic templates.C HA AP PT TE ER R 1 12 2 CH Related Products Comparison of Enzymes Used in PCR Product Taq DNA Polymerase Taq PCRX DNA Polymerase PCR SUPERMIX PLATINUM® Taq DNA Polymerase PLATINUM Taq PCRX DNA Polymerase PLATINUM PCR SUPERMIX PLATINUM Quantitative PCR SUPERMIX–UDG PLATINUM Pfx DNA Polymerase PCR SUPERMIX High Fidelity PLATINUM Taq DNA Polymerase High Fidelity PLATINUM GENOTYPE Tsp DNA Polymerase ELONGASE® Enzyme Mix * Contains PCRX Enhancer System.1 PLATINUM Taq DNA Polymerase*. 18038-018 18038-042 18038-067 10342-053 10342-020 10342-046 10966-018 10966-026 10966-034 11708-013 11708-021 11708-039 11304-022 11304-029 11508-017 11509-015 11448-024 11448-032 10965-010 10965-028 Size 100 units 500 units 1.500 units 100 units 250 units Taq DNA Polymerase.500 units 100 units 500 units 1. and MgSO4) PLATINUM Taq DNA Polymerase High Fidelity*.1 Cat.2. and MgCl2) PLATINUM GENOTYPE Tsp DNA Polymerase*. Recombinant*.2. Native* .14 Taq PCRX DNA Polymerase*.500 units (3 × 500 units) 100 units 250 units 500 units 100 units 250 units 500 units 100 units 500 units 500 units 500 units 250 units 2.14 22 R E L A T E D P R O D U C T S .5 kb <30 kb Yield Speciﬁcity Fidelity • • • •• •• •• •• •• •• ••• • •• • •* • •• ••* •• •• ••* • •• •• • • • • • • • • •••• ••• ••• • •• PCR Enzymes Product Taq DNA Polymerase.14 (Includes PLATINUM Pfx DNA Polymerase.
14.2.13. RNASEOUT RNase Inhibitor.14 PCR SUPERMIX High Fidelity*.2 ELONGASE Ampliﬁcation System*. 10572-014 11306-016 10790-020 11730-017 11730-025 10198-018 10481-018 10480-010 Size 100 reactions 100 reactions 100 reactions 100 reactions 500 reactions 100 reactions 100 reactions 100 reactions RT-PCR Product SUPERSCRIPT II RNase H Reverse Transcriptase ™ Cat.14 (Includes SUPERSCRIPT II RT/PLATINUM Taq DNA Polymerase Mix.C HA AP PT TE ER R 1 12 2 CH PCR Enzymes (continued) Product PCR SUPERMIX*. random hexamers. 5. coli RNase H) THERMOSCRIPT RT-PCR System and PLATINUM Taq DNA Polymerase*. No. No. E.2 Cat. 5. RNASEOUT™ RNase Inhibitor 10X RT buffer. Version 2.12.000 units 200. oligo(dT)20.000 units 50 reactions 18064-014 18064-022 18064-071 18053-017 11904-018 SUPERSCRIPT RNase H– Reverse Transcriptase 5 SUPERSCRIPT First-Strand Synthesis System for RT-PCR 5 (Includes oligo(dT)12-18. SUPERSCRIPT II RT.5 (Includes HeLa RNA and β-actin primers) R E L A T E D P R O D U C T S 23 . random hexamers. and MgSO4) THERMOSCRIPT™ RT-PCR System*. 2X reaction buffer.14 THERMOSCRIPT RT-PCR System and PLATINUM Taq DNA Polymerase High Fidelity*. – 5 Size 2. 5X cDNA Synthesis Buffer. Mg solution) 10928-018 10928-026 10928-034 10928-042 11922-010 11922-028 11146-024 11146-016 11146-057 11146-032 11146-040 11731-015 11731-023 28025-013 28025-021 10929-016 18021-014 18021-071 18068-015 10777-019 10813-012 18374-058 18373-019 25 reactions 100 reactions 25 reactions 100 reactions 25 reactions 100 reactions 25 reactions 100 reactions 25 reactions 100 reactions 100 reactions 100 reactions 500 reactions 40. control RNA and primers.13 (Includes THERMOSCRIPT RT.000 units 10. 2X Reaction Mix.000 units 10.2.24 (Includes THERMOSCRIPT PLUS/PLATINUM Taq Mix.5 3´ RACE System for Rapid Ampliﬁcation of cDNA End 4.25 ml 10 reactions 20 reactions SUPERSCRIPT One-Step RT-PCR System with PLATINUM Taq DNA Polymerase 2.14. and MgSO4) SUPERSCRIPT One-Step RT-PCR for Long Templates 2. and MgSO4) M-MLV Reverse Transcriptase RT-PCR Primer and Control Set Ribonuclease H DNase I. E.2.2.1 M DTT. 5 (Includes SUPERSCRIPT II RT/Taq DNA Polymerase enzyme mix. Ampliﬁcation Grade RNASEOUT Recombinant Ribonuclease Inhibitor DEPC-treated Water 5´ RACE System for Rapid Ampliﬁcation of cDNA Ends.13.000 units 20 reactions 30 units 120 units 100 units 5.2. DEPC-treated water.12. Mg solution.1.2. 2X THERMOSCRIPT Reaction Mix. and DEPC treated water) SUPERSCRIPT One-Step RT-PCR System*. 0.13.14 PLATINUM Quantitative RT-PCR THERMOSCRIPT One-Step System*.000 units 4 × 1. 10 mM dNTP Mix. 10 mM dNTP mix.12 PLATINUM Quantitative PCR SUPERMIX-UDG*. coli RNase H.2.12 PLATINUM PCR SUPERMIX*.0 4.000 units 4 × 10. 9.14 (Includes SUPERSCRIPT II RT/PLATINUM Taq DNA Polymerase High Fidelity Mix. 24 PCR Reagent System*.2.2 ELONGASE Enzyme Mix*.1. 2X Reaction Mix.2.
16 CONCERT™ Rapid PCR Puriﬁcation System CONCERT Rapid Gel Extraction System TAQUENCH™ PCR Cloning Enhancer 4. No. 15596-026 15596-018 10296-010 10296-028 10503-027 10503-035 10974-020 10974-038 10978-021 15513-039 25530-015 10786-010 10786-036 10876-019 11458-015 11458-023 11456-019 11456-027 11265-014 11265-022 Size 100 ml 200 ml 100 ml 200 ml 100 ml 200 ml 100 ml 200 ml 100 ml 100 ml 100 mg 100/pkg 100/pkg 250 ml 50 reactions 250 reactions 50 reactions 250 reactions 100 units 250 units Other Applications Product AFLP Analysis System I (Genome size of 5 × 10 to 6 × 10 ) ® Cat.C HA AP PT TE ER R 1 12 2 CH Related Products (continued) Reaction Components Product GIBCO BRL® Custom Primers 10 mM dNTP Nucleotide Mix 4 100 mM dNTP Nucleotide Set 4 Cat.16 FTA Puriﬁcation Reagent 4. No.16 FTA GeneCard 4. No.14 ® Cat.12. Please Inquire 18427-013 10297-018 Size 100 µl 4 × 250 µl Nucleic Acid Puriﬁcation Product TRIZOL Reagent TRIZOL LS Reagent DNAZOL® Reagent DNAZOL BD Reagent Plant DNAZOL Reagent Buffered-Saturated Phenol Proteinase K FTA® Cards 4. 8 9 17 Size each each each 125 µl 10544-013 10717-015 11352-010 10822-013 (Contains AFLP Core Reagent Kit and AFLP Starter Primer Kit) AFLP Analysis System II (Genome size of 1 × 108 to 5 × 108)17 (Contains AFLP Core Reagent Kit and AFLP Small Genome Primer Kit) AFLP System for Microorganisms (Designed for bacteria and fungi)17 (Contains AFLP Core Reagent Kit and AFLP Microorganism Primer Kit) AFLP Non-radioactive Probe 24 R E L A T E D P R O D U C T S .
com These products are for laboratory research use only and are not intended for human or animal diagnostic. Inc. ART®. AFLP® is a registered trademark of Keygene n. Inc. Roche Molecular Systems. FTA® is a registered trademark of Flinders Technologies PTY Ltd.. ABI PRISM® is a registered trademark of Perkin Elmer Corporation. and The Perkin-Elmer Corporation. SYBR® is a registered trademark of Molecular Probes..2-ml PCR Tubes 0. HOTSTART 100®.000 tubes/bag 1. Hoffmann-La Roche Ltd. Inc. CFLP™ is a trademark of Third Wave Technologies. TaqMan® is a registered trademark of Roche Molecular Systems.v.000 tubes/bag 1. Inc. and REACH™ are marks of Molecular Bio-Products.® Aerosol Resistant Tips™ .5-ml PCR Tubes 0. * Purchase of this product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for research and development in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee. 10331-015 10332-013 10467-017 10468-015 10890-010 18054-015 Size 96 tubes 96 tubes 96 tubes 96 tubes 4 ml 100 units ART. No. 15628-019 10787-018 10795-011 10795-037 10795-151 10795-144 Size 50 µg 250 µg 1. unless otherwise stated. Academic/TECH-LINE™: (800) 828-6686 U. R E L A T E D P R O D U C T S 25 . Industrial Orders/TECH-LINE: (800) 874-4226 Internet: www. Inc. therapeutic.lifetech. PfuTurbo™ is a trademark of Stratagene..e. Brand Pipet Tips Product ART REACH ART 20P ART 20E ART 200 ART 1000 ™ Cat.S. Inc.2-ml PCR Tubes with Flat Caps 0. HOTSTART Micro 20®. either by payment to Perkin-Elmer or as purchased. No. No.. an authorized thermal cycler. Aerosol Resistant Tips™. DNAZOL® and TRIZOL® are registered trademarks of Molecular Research Center. Inc. 10670-016 14862-015 14862-023 14867-014 14868-012 Size 960 tips/pkg 1.2-ml PCR Tubes with Attached Strip Caps Cat. Government Orders/TECH-LINE: (888) 584-8929 U.C HA AP PT TE ER R 1 12 2 CH Related PCR Products Product 100 bp DNA Ladder 1 Kb PLUS DNA LADDER™ 0. HOTSTART 50®.S. This product is sold under licensing arrangements with F. or other clinical uses. and HOTSTART Micro 50® are registered trademarks of Molecular Bio-Products.000 tubes/bag 125 strips/bag Thin-wall Reaction Tubes with Wax Beads Product HOTSTART 50 Storage and Reaction Tubes (20 to 50 µl reactions) HOTSTART 100® Storage and Reaction Tubes (50 to 100 µl reactions) HOTSTART Micro 20® Storage and Reaction Tubes (15 to 25 µl reactions) HOTSTART Micro 50® Storage and Reaction Tubes (25 to 50 µl reactions) Silicone Oil Uracil DNA Glycosylase 9 ® Cat.S.000 tips/pkg 960 tips/pkg 960 tips/pkg 800 tips/pkg U. i.
is willing to accept return of the products with a full refund.996. HoffmannLa Roche Ltd (“Roche”). MD 20850.683. 850 Lincoln Centre Drive. Alameda. 4. and this product may not be used in any manner other than provided herein. has an up-front fee component and a running-loyalty component... Rockville. or plants. whether or not such product or its components are resold for use in research. without prior written agreement between the buyer and Life Technologies. 1145 Atlantic Avenue.S. Further information on purchasing licenses to practice the PCR Process may be obtained by contacting the Director of Licensing at the Perkin-Elmer Corporation. For information on purchasing a license to this product for purposes other than research. or is available form The Perkin-Elmer Corporation.195. whether or not such stand-alone reagent is resold or such kit is resold for use in research. when used in conjunction with an Authorized Thermal Cycler. Inc.195. Alameda. Alameda. 9 The use of uracil-N-glycosylase for carryover prevention in ampliﬁcation reactions is the subject of claims in U. 1145 Atlantic Avenue. 9800 Medical Center Drive. Further on purchasing licenses to practice the PCR Process may be obtained by contacting the Director of Licensing at the Perkin-Elmer Corporation. California 94404 or at Roche Molecular Systems. Inc. owned by Hoffmann-La Roche Inc.C HA AP PT TE ER R 1 12 2 CH Related Products (continued) Limited Label Licenses 1 A license under U. Patents 5. Rockville. Rights to the up-front fee component must be obtained by the end user in order to have a complete license. 5 Purchase of this product conveys to the buyer only the non-transferable right to use the product in research conducted by the buyer.216 or their foreign counterparts. The purchase price of this product includes limited. Foster City. and 5. California 94404 or at Roche Molecular Systems. contact the Director of Licensing. No rights to use in other applications including the diagnosis of disease in humans. California 94501.965. Fax (301) 610-8383.683. Purchase of this product conveys the right to use it for research purposes only in processes claimed in the aforementioned patents. These rights under the up-front fee component may be purchased from Perkin-Elmer or obtained by purchasing an Authorized Thermal Cycler. 1145 Atlantic Avenue. 9800 Medical Center Drive. is willing to accept return of the product with a full refund. and F. For information on purchasing a license to this product for purposes other than research. 4. or to resell the product or components of the product as a standalone reagent or in a kit. owned by Hoffman-La Roche Inc. The buyer cannot sell or otherwise transfer this product to a third party. nontransferable rights under the running-royalty component to use only this amount of the product to practice the Polymerase Chain Reaction (“PCR”) and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. animals. Further information on purchasing licenses to practice the PCR Process may be obtained by contacting the Director of Licensing at the Perkin-Elmer Corporation. No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product. contact the Director of Licensing. 2 4 This product optimized for use in the Polymerase Chain Reaction (PCR) covered by patents owned by Hoffman-La Roche Inc. A license to use the PCR Process for certain research and development activities accompanies the purchase of certain reagents from licensed supplies such as Life Technologies. California 94404 or at Roche Molecular Systems. (1) use of the product or its components in manufacturing. These rights under the up-front fee component may be purchased from Perkin-Elmer or obtained by purchasing an Authorized Thermal Cycler.075. information or data. California 94501.. reporting the results of the purchaser’s activities for a fee or other commercial consideration. 5. (3) use of the product or its components for diagnostic purposes. including without limitation reporting the results of the purchaser’s activities for a fee or other commercial considerations. Inc.188. no rights are conveyed to the buyer to use the product in manufacturing. Foster City. Phone (301) 610-8000. 850 Lincoln Centre Drive. is hereby granted by implication or estoppel. Commercial purposes means any activity for which a party receives consideration and may include. and F.896. The purchase price of this product includes limited. reserves all other rights. Life Technologies. (2) use of the product or its components to provide a service. Inc. Inc. is hereby granted by implication or estoppel. without limitation. 850 Lincoln Centre Drive. or (4) resell the product or its components. Phone (301) 610-8000. Patent 4. Inc. Inc. has an up-front component and a running-royalty component. Life Technologies. Inc. If purchaser is not willing to accept the limitations of this limited use statement. Fax (301) 610-8383.638. California 94501. Inc. Hoffman.La Roche Ltd (“Roche”). Inc. no rights are conveyed to the buyer to use the product or its components for commercial purposes.683. The purchase of this product conveys to the buyer the non-transferable right to use the product and components of the product in research conducted by the buyer.035. nontransferable rights under the running-royalty component to us only this amount of the product to practice the polymerase chain reaction (PCR) and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. A license under U. Foster City.S.202.188 or their foreign counterparts.683. and 4. The buyer cannot sell or otherwise transfer this product or its components to a third party and in particular. 4. and foreign equivalents owned by Life Technologies. MD 20850. 12 26 R E L A T E D P R O D U C T S .202. Patents 4. No right to perform of offer commercial services of any kind using PCR including. but is not limited to. If the purchaser is not willing to accept the limitations of this Label License.. under any patents owned by Life Technologies. are conveyed by the purchase of this product.965. and F. In particular.S. Life Technologies. No right to perform of offer commercial services of any kind using PCR. Rights to the up-front fee component must be obtained by the end user in order to have a complete license. Hoffmann-La Roche Ltd (“Roche”).
Patent Nos. A license to practice this technology must be obtained from PE Biosystems. 9800 Medical Center Drive. and by U.S. (3) use of the product or its components for diagnostic purposes. Rockville.v.587. 6700 AE Wageningen. 5..834. and foreign equivalents for laboratory research and human identity testing. (2) use of the product or its components to provide a service. Patents 5. CA 94404. or (4) resell the product or its components. Material supplied by Life Technologies. contact Life Technologies. including but not limited to the use for clinical.671.S.S.538. Inc. is not intended for human or animal diagnostic or therapeutic uses.015 and 5. 5. and foreign equivalents for use in research only. or from Roche Molecular Systems.562. The Netherlands. Rockville. This kit may be used for research purposes only. owned by PE Corporation. and foreign equivalents. MD. whether or not such product or its components are resold for use in research.217 and 5. under U.847. 9800 Medical Center Drive.972. Phone (301) 610-8000. Inc. Forest City. 5.487.. under U. 5..126. (1) use of the product or its components in manufacturing. Fax (301) 610-8383.636. This product is sold under license from Keygene n. information. purchase of this product does not convey to the purchaser a license to practice any claim of any patent. P. contact the Director of Licensing. Patents 5. owned by Roche Molecular Systems. Inc. The use of TaqMan® ﬂuorogenic probes in 5´ nuclease assays is covered by U. Purchase of the product does not provide a license to use this patented technology. Inc.. Inc. Purchase of this product conveys to the buyer only the non-transferable right to use the product for laboratory research and non-commercial human identity testing. requires a license from Keygene n. For information on purchasing a license to this product for purposes other than research. Life Technologies. MD 20850. Fax (301) 610-8383. MN.075.. and/or therapeutic purposes.496. diagnostic. For information concerning the availability of product and licenses to practice the patented technology for clinical diagnosis or therapeutic uses contact FITZCO. Inc. Maple Plain.338. Commercial purposes means any activity for which a party receives consideration and may include. or for providing services to third parties. 15 Unless indicated otherwise.v.O. Inc. Use of this product for any commercial purposes or alteration of FTA Cards and related products in any manner is prohibited without the prior written consent of Life Technologies.. whether commercial or noncommercial. contact the Director of Licensing. Inc. Patent 5.807. The buyer cannot sell or otherwise transfer this product or its components for commercial purposes. Users of this product should determine if any license is required under these or any other patents. CA 94501. Patent No. The use of this kit for any other purpose. The AFLP technique is covered by patents or patented applications owned by Keygene n. including but not limited to U.756. For use of this kit in plant breeding. Inc. Licensed to Life Technologies.v. 1145 Atlantic Avenue. For information concerning the availability of product and licenses to practice the patented technology for commercial purposes in human identity testing. but is not limited to.848. 5.S. 16 R E L A T E D P R O D U C T S 27 . is willing to accept return of the products with a full refund. German Patent 3. Box 216.S. Alameda. If purchaser is not willing to accept the limitations of this limited use statement.766. Rockville. Life Technologies. 17 24 14 Licensed to Life Technologies. Phone (301) 610-8000. Inc. MD 20850.210.C HA AP PT TE ER R 1 12 2 CH 13 Purchase of this product conveys to the buyer the non-transferable right to use the product and components of the product in research conducted by the buyer. 850 Lincoln Center Drive.527.287. or data.
76 18 18 18 18 — Primers do not amplify pseudogenes.8 Reference 47 47 47 28 S E L E C T E D P R I M E R S E Q U E N C E S .62 0.11 4.5 Reference 70 70 71.5 5.72 73.74 75.1 2.3 1.49 0.8 3.50 0.20 12.3 6.A PP PE EN ND DI IX X A A AP Selected Primer Sequences RT-PCR Primer Sequences Gene β-actin β-actinA β-actin GAPDH GAPDH Dynein Polymerase ε Polymerase ε Tuberous Sclerosis 18S rRNA A Source Human Rat Mouse Human Rat Rat Human Human Human Soybean Primer sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense Sequence CCTCG CCTTT GCCGA TCC GGATC TTCAT GAGGT AGTCA GTC TACAA CCTCC TTGCA GCTCC GGATC TTCAT GAGGT AGTCA GTC GTCGT ACCAC AGGCA TTGTG ATGG GCAAT GCCTG GGTAC ATGGT GG GGTGA AGGTC GGAGT CAACG CAAAG TTGTC ATGGA TGACC GATGC TGGTG CTGAG TATGT CG GTGGT GCAGG ATGCA TTGCT CTGA GCGGG CGCTG GAGGA GAA GGATC TTCAT GAGGT AGTCA GTC CGCCA AATTT CTCCC CTGAA CCGTA GTGCT GGGCA ATGTT C AAGGC TGGCG GATTA CTGCC GATGC TGCTG GTGAT GTACT C GGAGT TTATC ATCAC CGCGG AAATA CTGAG AG TATTT CACTG ACAGG CAATA CCGTC CAAGG CTTTC GATGG TAGGA TAGTG GCCT CAATG ATCCT TCCGC AGGTT CACCT AC Product Size (kb) 0.62 0. PCR Primer Sequences Gene HIV gag region β-globin β-globin Source Viral Human Human Primer SK 38 SK 39 29923 34016 31194 34016 Sequence ATAAT CACTA TCCAG TAGGA GAAAT TTTGG TCCTG TCTTA TGTCC AGAAT GC GGTGT TCCCT TGATG TAGCA CA CCAGG ATTTT TGATG GGACA CG GCTGC TCTGT GCATC CGAGT GG CCAGG ATTTT TGATG GGACA CG Product Size (kb) 0.
DNase-RNase-Free ART Tips PCR Tubes HOTSTART Storage and Reaction Tubes Silicone Oil MICROMATE Labels Electrophoretic Analysis of PCR Products 25.PCR and RT-PCR Product Groupings Plasmid DNA Isolation CONCERT Rapid Plasmid Puriﬁcation System CONCERT High Purity Plasmid Puriﬁcation System Genomic DNA Isolation DNAZOL Reagents FTA Cards and Reagent Phenol Products PRETAQ Thermophilic Protease Proteinase K RNA Isolation TRIZOL Reagents MESSAGEMAKER System GLASSMAX RNA System RNASEOUT Ribonuclease Inhibitor Proteinase K RNA Molecular Size Standards DEPC-treated Water First-Strand cDNA Synthesis (RT-PCR) SUPERSCRIPT First-Strand Synthesis RT-PCR System SUPERSCRIPT One-Step RT-PCR Systems THERMOSCRIPT RT-PCR Systems PLATINUM Quantitative RT-PCR THERMOSCRIPT System 3´ RACE System 5´ RACE System Custom Primers DNAse I. 50. 250 EX. 250. and 123 bp DNA Ladders 1 Kb PLUS DNA Ladder HORIZON Electrophoresis Apparatus SUNRISE Electrophoresis Apparatus Models 250. and 125 Power Supply Electrophoresis Reagents Agarose Agarose 1000 10X BLUEJUICE Buffer TBE Buffer TAE Buffer Ethidium Bromide Kodak Digital Science EDAS Systems Cloning PCR Products CONCERT Rapid PCR Puriﬁcation System TAQUENCH Cloning Enhancer Restriction Endonucleases T4 DNA Ligase Competent Cells CLONEAMP Systems PCR Cloning System with GATEWAY Technology P C R A N D R T . Ampliﬁcation Grade dNTPs Ribonuclease H M-MLV RT SUPERSCRIPT II Reverse Transcriptase RT-PCR Primer and Control Set PCR Enzymes including: Taq DNA Polymerase PLATINUM Enzymes (high speciﬁcity) PLATINUM Pfx DNA Polymerase (high ﬁdelity) PLATINUM GENOTYPE Tsp DNA Polymerase High Fidelity Enzyme Mixes PCR x DNA Polymerases (high GC content) SUPERMIXES PLATINUM Quantitative PCR SUPERMIX-UDG ELONGASE Reagents PCR Reagent System Custom Primers dNTPs Uracil DNA Glycosylase Distilled Water. 100.P C R P R O D U C T G R O U P I N G S 29 . and 500 bp DNA Ladders 10.
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