PCR Basics

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Purpose of PCR Overview Components of PCR Reaction Variables
Temperature Cycle Times and Numbers Primer Buffer Polymerase

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Experimental Notes

Polymerase Chain Reaction
‡ ³Amplify´ large quantities of DNA ( g quantities) from small quantities (fg quantities) [billion fold amplification] ‡ Analyze single DNA fragments out of large complex mixture. [ Human genome mixture of 12 million 300bp fragments] ‡ Alter DNA sequence ± directed mutagenesis.

What is the Polymerase Chain Reaction?
‡ It¶s a means of selectively amplifying a particular segment of DNA. ‡ The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene. ‡ It can be thought of as a molecular photocopier

How Powerful is PCR?
‡ PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. ‡ The template DNA need not be highly purified ² a boiled bacterial colony. ‡ The PCR product can be digested with restriction enzymes, sequenced or cloned. ‡ PCR can amplify a single DNA molecule, e.g. from a single sperm.

The Invention of PCR

Did He Really Invent PCR? ‡ The basic principle of replicating a piece of DNA using two primers had already been ‡ described by Gobind Khorana in 1971: ± Kleppe et al. . 56. Mol. 341346. (1971) J. ‡ Mullis properly exploited amplification. ‡ Progress was limited by primer synthesis and polymerase purification issues. Biol.

± extension (72°C). time depends on product size. ± annealing (55±60°C). 30 sec. 30 sec. What¶s in the Reaction? .The Basics of PCR Cycling ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ 30±35 cycles each comprising: ± denaturation (95°C).

ammonium ions (and/or potassium ions). magnesium ions. ‡ bovine serum albumin) ‡ Nucleotides (dNTPs) ‡ Primers ‡ DNA polymerase (usually Taq) .WHAT¶S IN THE REACTION ? ‡ Template DNA ‡ Reaction buffer (Tris.

PCR In Detail ‡ Denature. extend and repeat the cycle 30 to 35 times. ‡ ³How does the polymerase know to stop when it reaches the other primer?´ ‡ Most textbooks to not fully explain PCR. . anneal.

‡ The accumulation is not strictly a doubling at each cycle in the early phase. ‡ There are also 60 other DNA copies . ‡ At 30 cycles there are 1.How many copies? ‡ No target products are made until the third cycle.764 target copies (~1×109).741.073.

Temperature Cycling Denaturation Annealing Extension 94° 55° 72° 2.Overview of PCR 1. Every cycle DNA between primers is duplicated .

PCR Amplification .

1 billion copies in theory .Exponential Amplification 30 cycles --.

dCTP. dGTP) ‡ PCR Buffer (mg++) ‡ Thermocyler . dTTP.Components of PCR Reaction ‡ Template DNA ‡ Flanking Primers ‡ Thermo-stable polymerase ± Taq Polymerase Thermus aquaticus ‡ dNTP ± (dATP.

5. Temperature Cycle Times and Temps Primer Buffer Polymerase . 2. 3. 4.PCR Variables 1.

Temperature ‡ Denaturation ± Trade off between denaturing DNA and not denaturing Taq Polymerase ‡ Taq half-life 40min at 95 °.5° ± 95° ‡ Annealing ± Trade off between efficient annealling and specificity ± 2-5 ° below Tm ‡ Extension ± Temperature optimum for Taq Polymerase ± 72 ° . 10min at 97.

Cycle Times and Temps Typical PCR Run Step Time/Temp 1 2 3 4 5 6 7 8 3 min at 95° 30 sec at 95° 1 min at 55° 2 min at 72° Go to step 2 .29 times 8 min 72° 0 min 4° End .

Unwanted products accumulate . ‡ The plateau occurs when: ± The reagents are depleted ± The products re-anneal ± The polymerase is damaged .How many cycles? ‡ Increasing the cycle number above ~35 has little positive effect.

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heterodimers . hairpins.Primers ‡ ‡ ‡ ‡ ‡ ‡ ‡ Paired flanking primers Length (17-28bp) GC content 50-60% GC Clamp Tm¶s between 55-80 Avoid simple sequences ± e.g. strings of G¶s Avoid primer self complementary ± e.g. homodimers.

primers and DNA templates. ‡ ‡ ‡ ‡ dimethyl sulphoxide (DMSO). dimethyl formamide (DMF). and too many increase the yield of non-specific products and promote misincorporation.useful when target DNA is high G/CWith NAs of high (G+C) content. Too few Mg2+ ions result in a low yield of PCR product.5 mM MgCl2 ‡ ‡ Magnesium ± Since Mg ions form complexes with dNTPs. urea formamide ± Long Targets >1kb.PCR Buffer ‡ Basic Components ± 20mM Tris-HCL pH 8. Formamide and glycerol ± Low concentration of template: Polyethylene glycol (PEG) . the optimal concentration of MgCl2 has to be selected for each experiment.4 ± 50mM KCl ± 1. Potential Additives ± Helix Destabilisers .

Vent 7 hour half-life ‡ 3¶-5¶ Exo nuclease ± proofreading ‡ Fidelity (Error Rate) ± Taq 1/10. Pfu 1/1.PCR Polymerases ‡ Taq.g. Pfu.000 ‡ Processivity ‡ Extra bases at end . others ‡ Native or Cloned ‡ Half-life ± e.000. Vent. Taq 40 min half-life.000nt.

Notes ‡ Typical Reaction ‡ 32.5 l 50 l dH2O 10 X PCR buffer + mg 200 M dNTP 50 M Left Primer 50 M Right Primer Worm or Fly Lysate Taq Pol (5 Units/ l) Total Vol .5 l ‡ 5 l ‡ 1 l ‡ 0.5 l ‡ 10 l ‡ 0.5 l ‡ 0.

5 l Right primer .4 l 200 M dNTP ± 2.10 l worm lysate ‡ ‡ .5 l Left primer .0.Master Mix ‡ 1 volume master mix ± 32.5 l Taq Pol (5 Units/ l) 39 l Total Volume To set up 4 reactions prepare 4.2 l Taq Pol (5 Units/ l) Individual reactions .4 volumes of reaction master mix ± 143 l dH2O ± 22 l 10 X PCR buffer ± 4.39 l master mix .0.5 l dH2O ± 5 l 10 X PCR buffer ± 1 l 200 M dNTP ± 0.

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DNA Sequencing .

Automated method 2. Sanger dideoxynucleotide chain termination method (Commonly used method) A. Manual method B. Chemical cleavage method (Maxam and Gilbert method) Not used nowadays Use of the technique: Provides the order of the nucleotides in a given DNA .DNA Sequencing Two main methods 1.

Sanger Method .DNA Sequencing .

Sanger Method .

DNA Sequencing .Sanger Method .

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adjacent to a primer site ‡ Four reactions are set up -.Procedure for the Sanger dideoxy chain termination technique DNA to be sequenced is cloned into a vector. giving rise to sets of DNA products representing all of the possible size fragmentsfor the unknown sequence ‡ The fragments are resolved by gel electrophoresis and the sequence is read up from the bottom of the gel by identifying the lane giving the next larger size fragment .each containing one of four ddNTPs ‡ DNA is synthesized in the presence of the ddNTPs.

dTTP ‡ One type of ddNTP in each type of reaction ‡ DNA polymerase (Taq polymerase can also be usedcycle sequencing) ‡ One primer ‡ Template DNA ‡ Labeling.Composition of a DNA sequencing reaction ‡ dNTP ± dATP.Radioactive and non radioactive . dGTP. dCTP.

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Labeling the primers S35dNTP or P32 dNTP Or Fluorescent labeled dNTP . Labeling the nucleotides S35dNTP or P32 dNTP Or Fluorescent labeled dNTP 2.Labeling methods 1.

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For sequencing ‡ the DNA is denatured into single strands ‡ the primer is hybridized to the template strand ‡ DNA is synthesized using DNA polymerase .

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