PCR and its application

Continued..

Ref: Cloning Heterologous Genes: Problems and Approaches

Jacqueline Agnan, Christopher Korch and Claude Selitrennikoff
Fungal Genetics and Biology 21, 292–301 (1997) ,Article No. FG970995 Wikipedia and cross references

Thermostable Polymerases
• Taq: Thermus aquaticus (most commonly used)
– Sequenase: T. aquaticus YT-1 – Restorase (Taq + repair enzyme)

• • • • • •

Tfl: T. flavus Tth: T. thermophilus HB-8 Tli: Thermococcus litoralis Carboysothermus hydrenoformans P. kodakaraensis (Thermococcus) Pfu: Pyrococcus furiosus

Thermostable Polymerases
Polymerase Taq pol Amplitaq (Stoffel fragment) Vent Deep Vent Pfu Tth T ½, 95oC 40 min 80 min Extension Type of Rate (nt/sec) ends 75 3’A >50 3’A Source T. aquaticus T. aquaticus

400 min 1380 min >120 min 20 min

>80 ? 60 >33

95% blunt 95% blunt Blunt 3’A

Thermococcus litoralis Pyrococcus GB-D Pyrococcus furiosus T. thermophilus

APPLICATION

APPLICATION OF PCR &

VARIENTS OF PCR

Analysis of PCR Products
Analysis of Amplicons
Restriction Digestion Electrophoresis

Cloning Electrophoresis DNA Sequence

Southern Electrophoresis
Fragment Size Transfer

Amplicon
Size

Analysis

Hybridize
with Probe

Detection

Application in Genetic Engineering .

Gene cloning  Cloning by PCR. DNA is stored for long time. DNA is amplified to analyze it  Cloning by plasmid followed by transformation. so does the DNA fragment . the plasmid gets copied and therefore. Bacteria divide and reproduce.

 Bases at the 5'-end of the primer are less critical for primer annealing. to the 5'-end of the primer molecule. like restriction sites. The first step is the design of the necessary primers  Not to have: 3'-end : 3 of more G or C bases at this position (this may stabilize nonspecific annealing of the primer). This often leads to primer-dimer formation.Cloning . a 3' thymidine (it is more prone to mispriming than the other nucleotides). Tm .PCR Strategy The gene of interest usually has to be amplified from genomic or vector DNA by before it can be cloned into an expression vector. GC content. Therefore.  Primer pairs should be checked for complementarity at the 3'-end.  Primer length. it is possible to add sequence elements.

Cloning Heterologous Genes  Many economically and medically important fungi are genetically less well characterized.  Cloned genes from model organisms are often used to identify and isolate the cognate genes from these fungi on the basis of assumed conservation of sequence and function . which makes it difficult to study their genes and gene products.

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which can be the basis for PCR amplification and cloning of a genomic or cDNA segment of a cognate gene of unknown sequence  The amplified segment can subsequently be used as a homologous probe to screen a DNA library . Heterologous probes may not always detect a cognate gene  The availability databases its possible to identify conserved regions of genes.

 Amplification of a DNA segment of unknown sequence can be done by using oligonucleotides of either specific or degenerate sequence  If the model gene has been sequenced „„in-house.  Designing primers that amplify a specific DNA sequence from a heterologous organism can be a challenging task. . Obtaining a homologous product by PCR is not always simple.‟‟ it is worthwhile to try the sequencing primers first.  Successful amplification of a DNA segment of unknown sequence by PCR depends primarily on the sequence of the primers.

as several different codons can code for one amino acid.Degenerate primers These are actually mixtures of similar. The problem can be partly solved by using touchdown PCR . thymine. as the genes themselves are probably similar but not identical The other use for degenerate primers is when primer design is based on protein sequence. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. where H for adenine. it is often difficult to deduce which codon is used in a particular case Isoleucine might be "ATH". They may be convenient if the same gene is to be amplified from different organisms. or cytosine. but not identical primers.

and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles .  The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used. while at the later cycles.TOUCH DOWN PCR (Step-down PCR):  A variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses.  The higher temperatures give greater specificity for primer binding. it is a few degrees (3-5°C) below the primer Tm.

Overlap extension polymerase chain reaction For splicing Deleting a portion Induction of mutation .

Splicing by Overlap Extension PCR Joining two DNA sequences .

Deleting a DNA sequences .

DNA Ploymorphism .

Amplicon size  For genetic diversity/ relation  For identification of individual  Marker  Genetic fidelity .

DNA Polymorphism .

dominant SSR – co-dominant .dominant AFLP .dominant ISSR .Some of the conventional PCR based markers RAPD .

Genetic fidelity .

PCR Amplification of Specific Alleles (PASA) Forward for G allele: 5‟-CCTGGACAACATGGTGACACTCC-3‟ Forward for C allele: 5‟-CCTGGACAACATGGTGACACTCG-3‟ Reverse common: 5‟-GTGGGCCCTGTACTTTTACATC-3‟ .Allele-specific (AS-) PCR: A diagnostic or cloning technique based on single-nucleotide polymorphisms (SNPs) . and uses primers whose 3' ends encompass the SNP. It requires prior knowledge of a DNA sequence. including differences between alleles.

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.DNA Fingerprinting Identification of individual Criminal identification – Forensic Sc. Parentage Plant/Animal/Species identification Seed material etc.

Diagnostic PCR Amplification From Patient Samples 104 bp .

 Annealing temperatures for each of the primer sets must be optimized .  A single test-run that otherwise would require several times the reagents and more time to perform.Multiplex-PCR  Consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences.

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Asymmetric PCR  Preferentially amplifies one DNA strand in a double-stranded DNA template  It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required  PCR is carried out as usual. extra cycles of PCR are required . but with a great excess of the primer for the strand targeted for amplification  Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up.

DNA Sequencing .

Chain Termination (Sanger) Sequencing • A modified DNA replication reaction. • Growing chains are terminated by dideoxy nucleotides. .

Chain terminates at ddG .Chain Termination (Sanger) Sequencing • The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs.

Primer 5′OPTemplate -3′ OH TCGACGGGC… Template area to be sequenced • Dideoxy nucleotides are added separately to each of the four tubes.Chain Termination (Sanger) Sequencing • A sequencing reaction mix includes labeled primer and template. .

Chain Termination (Sanger) Sequencing A ddATP + four dNTPs ddCTP + four dNTPs ddA dAdGdCdTdGdCdCdCdG dAdGddC dAdGdCdTdGddC dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC dAddG dAdGdCdTddG dAdGdCdTdGdCdCdCddG C G ddGTP + four dNTPs T ddTTP + four dNTPs dAdGdCddT dAdGdCdTdGdCdCdCdG .

Chain Termination (Sanger) Sequencing • With addition of enzyme (DNA polymerase). • With the proper dNTP:ddNTP ratio. • The chain will end with the incorporation of the ddNTP. the primer is extended until a ddNTP is encountered. the chain will terminate throughout the length of the template. • All terminated chains will end in the ddNTP added to that reaction. .

• Fragments from each of the four tubes are placed in four separate gel lanes. .Chain Termination (Sanger) Sequencing • The collection of fragments is a sequencing ladder. • The resulting terminated chains are resolved by electrophoresis.

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Sequencing: Classical Sequencing Gel .

• Cycle sequencing requires a heat-stable DNA polymerase.Cycle Sequencing • Cycle sequencing is chain termination sequencing performed in a thermal cycler. .

. • For sequencing applications. these molecules can be covalently attached to nucleotides. • Examples are fluorescein and rhodamine derivatives.Fluorescent Dyes • Fluorescent dyes are multi-cyclic molecules that absorb and emit fluorescent light at specific wavelengths.

the primer contains fluorescent dye–conjugated nucleotides. ddA ddA . labeling the sequencing ladder at the 3′ ends of the chains. the fluorescent dye molecules are covalently attached to the dideoxy nucleotides. labeling the sequencing ladder at the 5′ ends of the chains. • In dye terminator sequencing.Fluorescent Dyes • In dye primer sequencing.

A T AC G T GT The fragments are distinguished by size and “color.” . all four reactions can be performed in a single tube.Dye Terminator Sequencing • A distinct dye or “color” is used for each of the four ddNTP. • Since the terminating nucleotides can be distinguished by color.

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AT A GLANCE .

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Sequencing: .

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) • Steps in the RT-PCR reaction: – – – – RNA isolation Reverse transcription PCR amplification Analysis of PCR product .

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Reverse Transcription of RNA (RT) • Primer Options for RT reaction – Oligo (dT) – Random Hexamers – Sequence-specific Primers • Enzyme Options for RT Reaction – Retroviral RNA-directed DNA polymerase – AMV Reverse Transcriptase (avian myeloblastosis virus) – MMLV Reverse Transcriptase (Moloney murine leukemia virus) .

Real Time PCR (RT-PCR)
qPCR
 A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye.  An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified.  However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer).

Fluorescent reporter probe method

LATE-PCR
A recent modification on this process, known as Linear-After-TheExponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction

Smiths Detection has an exclusive license for LATE-PCR from Brandeis University for all markets, worldwide This enables one to detect low numbers of target organisms

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but takes advantage of DNA hybridization and annealing as well as DNA polymerase to amplify a complete sequence of DNA in a precise order based on the single stranded oligonucleotides used in the process.Assembly PCR / Polymerase cycling assembly ( PCA)  A method for the assembly of large DNA oligonucleotides from shorter fragments.  It thus allows for the production of synthetic genes and even entire synthetic genomes. .  The process uses the same technology as PCR.

The oligonucleotides alternate between sense and antisense directions. thereby selectively producing the final long DNA product . and the overlapping segments determine the order of the PCR fragments.

A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR .Dial-out PCR: a highly parallel method for retrieving accurate DNA molecules for gene synthesis.

Isolation of a gene by PCR If the Primers anneals anneal both the sides of the gene of interest No selection required Then why cloning Limitation: Sequence information ids required Length of DNA sequence If seq information is not known If we have information on heterologous gene / equivalent gene .

Its important to isolate gene whose seq is known Fish out gene .based on flanking sequence information Allelic forms of genes can be isolated Diagnostics – both in plant and animal including human Early diagnosis .

Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination. . but an adjunct. and to distinguish homologous recombination events from nonhomologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement. This is not a substitute for Southern blotting.

hence the name • This used to be the only way to amplify DNA. and make lots of bacterial cells that contain an identical molecule. • These cells are clones.Cloning • Cloning is the way in which we can take a single molecule. . It is still by far the most accurate.

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autonomous bacterial DNA .Plasmid vectors – circular.

The vector is made with a “T” overhang .

Only Taq polymerase leaves „A‟ overhangs. we need a „T‟ overhang. they leave “blunt” ends. it always puts an extra “A” at the end • This can be useful. . but note: other polymerases do not do this.Taq polymerase leaves an “A” overhang • Taq is the thermostable DNA polymerase from Thermus aquaticus we used for PCR. • When Taq synthesizes a new strand. „Blunt‟ end vectors do not work with Taq.

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DNA ligase • Repairs gaps in the sugar-phosphate backbone of DNA • Creates phosphodiester bonds • Does not do anything with the bases .

this method gets DNA into the cell by making them porous using CaCl2 and a 42 C heat treatment • Electroporation Makes cells porous using high-voltage electricity .Transformation of bacteria • Two main methods for transformation • Chemical / Heat Shock As done in last practical.

.Imperfect science • Most of the plasmid / insert combinations will not ligate • Most of the bacteria will not be transformed • We only need one molecule to get into one bacterium to make one colony.

..PCR from clones • Often clones will religate containing any old DNA (eg primer dimers). • The DNA can go in in either orientation • We can use the PCR to tell which colonies have the insert we want. and which orientation it is in.

Touch down PCR Touchdown PCR involves decreasing the annealling temperature by 1 degree C every second cycle to a 'touchdown' annealing temp which is then used for 10 or so cycles It was originally intended to bypass more complicated optimization processes for determining optimal annealing temperatures. . The idea is that any differences in Tm between correct and incorrect annealing gives a 2-fold difference in product amount per cycle (4fold per degree C). You therefore enrich for the correct product over any incorrect products.

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DD-PCR To detect the differential expression of the gene Usually subtractive-hybridization and differential cDNA cloning require large amounts of mRNA derived from cell or tissue samples. often taking many months from start to finish. . and are very laborious and time consuming. Alternatively Differential Display – PCR: requires only small amounts of cell or tissue samples. and can be completed within weeks.

N = any nucleotide) .Requires RNA derived from control (normal) and sample (diseased) cells or tissues The samples of RNA being examined are converted into cDNA by reverse transcription using an oligo-dT primer with two penultimate specific bases to the 3’ end 5’ TTTTTTTTTTTTMN 3’ (where M = A. G. or C.

different sets of random primers may be used After ~30 amplification cycles using radioactive S-35 labeled nucleotides the expressed cDNA fragments are displayed using DNA sequencing gels . and the same 3’ primer (shown above) In order to define most of the genes expressed in these cells.Upon conversion to cDNA the PCR amplification is done with arbitrary 5’ primer (8-10mers) using random sequences.

sequencing and data-base searches To verify the differential expression status of each of these sub-clones. and reamplified by PCR using the original primers. Northern-Blot assays are performed using RNA isolated from the original sample sources . The amplified DNAs are characterized by sub-cloning.Differentially expressed cDNAs are easily visualized following autoradiography The DNA from these gels slices is recovered by boiling.

COLONY PCR Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination. It can also be used to identify colonies from a tetrad that carry a particular gene replacement . and to distinguish homologous recombination events from nonhomologous.

MULTIPLEX PCR Two or more sets of primers. designed for amplification of multiple target Multiple targets can be detected from a single specimen in one reaction More complicated and less sensetive .

TRANSCRIPTION MEDIATED AMPLIFICATION OR NUCLEIC ACID SEQUENCE BASED AMPLIFICATION Isothermal amplification techniques RNA to DNA and DNA as a template for multiple copies of RNA (Like the life cycle of retrovirus) .

TMA/ NASBA Detection of HIV. rhinivirus. M. HCV. cytomegalovirus. measles. papillomavirus. vericella zoster virus. tuberculosis. M. pneumoniae etc. .

trachomatis.STRAND DISPLACEMENT AMPLIFICATION Isothermal amplification technique To detect trace amount od DNA or RNA of a particular sequence Detection of tuberculosis. gonorrhoea .

STRAND DISPLACEMENT AMPLIFICATION .

SDA .

gonorrhoeae etc.LIGASE CHAIN REACTION Two oligonucleotide probe hybridise side by side A thermostable DNA ligase seal the nick Each ligated product as well as original target serve as a template A modification of this technique – gapped LCR (G-LCR) Kits are available for the detection of C. . N.trachomatis.

LCR .

Denaturation at 94°C 2. M13. Annealing at 50°C 1.SIMPLE METHOD TO SEQUENCE LAMBDA. OR PLASMID CLONES USING PCR Three major steps 1. extension at 60°C .

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Detection .

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SEQUENCING .

G. PCR Protocols: A guide to methods and applications. A. CSHL press.. New York. M. D. H. H. Erlich. 1992. (1995) PCR primer: a laboratory manual. A. Gyllensten. Gelfand. Sninsky. 1989. 1991. C. Cold Spring Harbor. Mass. PCR technology: Principles and applications for DNA amplification. M. H. Atlas. Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Mahbubani. Eaton Publishing Co. Ellingboe. New York. and T. B. K. A.W and Dvksler. 1990.S. J. and R. Academic Press.. J. and U. The PCR technique: DNA sequencing.REFERENCE BOOKS Dieffenbach. USA.. Stockton Press. M. J.. . White. Natick. Critical Reviews in Biochemistry and Molecular Biology 26:301-334. Innis. Bej. J.

Denature (heat to 95oC) Lower temperature to 56oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs .

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DNA copies vs Cycle number 2500000 2000000 DNA copies 1500000 1000000 500000 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Cycle number .

Applications of PCR • Classification of organisms • Genotyping • Molecular archaeology • Mutagenesis • Mutation detection • Sequencing • Cancer research • Detection of pathogens • DNA fingerprinting • Drug discovery • Genetic matching • Genetic engineering • Pre-natal diagnosis .

Applications of PCR Basic Research • Mutation screening • Drug discovery • Classification of organisms • Genotyping • Molecular Archaeology • Molecular Epidemiology • Molecular Ecology • Bioinformatics • Genomic cloning • Site-directed mutagenesis • Gene expression studies Applied Research • Genetic matching • Detection of pathogens • Pre-natal diagnosis • DNA fingerprinting • Gene therapy .

Applications of PCR Molecular Identification • Molecular Archaeology Sequencing • Bioinformatics • Genomic cloning • Human Genome Project Genetic Engineering • Site-directed mutagenesis • Molecular Epidemiology • Molecular Ecology • DNA fingerprinting • Classification of organisms • Gene expression studies • Genotyping • Pre-natal diagnosis • Mutation screening • Drug discovery • Genetic matching • Detection of pathogens .

MOLECULAR IDENTIFICATION: .

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Molecular Identification: Detection of Unknown Mutations .

SSCP gels: “shifts” representing a mutation in the amplified DNA fragment .

Molecular Identification: Classification of Organisms 1) Relating to each other * Fossils 2) Similarities 3) Differences * Trace amounts * Small organisms Insufficient data ! DNA ! .

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Rademaker et al. 2001 .

Molecular Identification: Detection Of Pathogens .

Molecular Identification: Detection Of Pathogens Sensitivity of detection of PCR-amplified M.1994) . tuberculosis DNA. (Kaul et al.

(Kaul et al. tuberculosis DNA.Sensitivity of detection of PCR-amplified M.1994) .

Molecular Identification: Genotyping by STR markers .

Molecular Identification: Prenatal Diagnosis • Chorionic Villus • Amniotic Fluid 644 bp 440 bp 204 bp Molecular analysis of a family with an autosomal recessive disease. .

DNA sequencing – Sanger’s method .

SEQUENCING Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP) NO polymerisation after a dideoxynucleotide! Fragments of DNA differing only by one nucleotide are generated Nucleotides are either or .

Sequencing: Reading Classical Sequencing Gels .

hair root. Restriction Digestion.719. saliva 68.Summary blood.736 copies Gel Analysis. amniotic fluid. chorionic villus. Sequencing .476. semen.

.Conclusion The speed and ease of use. sensitivity. specificity and robustness of PCR has revolutionised molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications.

Brown. T. Blackwell .A.Gene Cloning and DNA analysis: An Introduction 6th ed.