Slide 1:Polymerase Chain Reaction Raj Kumar Duary Molecular biology unit, Dairy microbiology Division, National Dairy Research Institute Karnal, India-132001 E-mail : rkduary@gmail.com


Specific synthesis of DNA in vitro via a PCR reaction. (Emeryville; California, Cetus) :Specific synthesis of DNA in vitro via a PCR reaction. (Emeryville; California, Cetus) Invented by- Kary Mullis (1983) Shared the Nobel Prize in Chemistry with Michael Smith in 1993.


Slide 3:PCR is a rapid, inexpensive and simple way of copying specific DNA fragments from minute quantities of source DNA material • It does not necessarily require the use of radioisotopes or toxic chemicals • It involves preparing the sample DNA and a master mix with primers, followed by detecting reaction products


Slide 4:Chain reaction relies on DNA replication process Repeated cycles of melting (strand separation), primer annealing, and primer extension by cycling temperatures Need a tough enzyme to deal with high temperatures Polymerases isolated from thermophilic bacteria (Thermus aquaticus, Pyrococcus furiosus)


Slide 5:! Denaturation: DNA fragments are heated at high temperatures, which reduce the DNA double helix to single strands. These strands become accessible to primers ! Annealing: The reaction mixture is cooled down. Primers anneal to the complementary regions in the DNA template strands, and double strands are formed again between primers and complementary sequences ! Extension: The DNA polymerase synthesizes a complementary strand. The enzyme reads the opposing strand sequence and extends the primers by adding nucleotides in the order in which they can pair. The whole process is repeated over and over PCR procedures: steps


Slide 6:Melting temperature Tm°C = 2(A/T) + 4(G/C) Tm oC Temperature at which half possible H bonds are formed


Slide 7:The Thermus aquaticus DNA polymerase (Taq) Not permanently destroyed at 94ºC Optimal temperature is 72ºC


Does not have proof reading ability Error rate 1 in 2 X 104 bases Seems rare but can be recovered in cloning a single molecule Newer polymerases have high fidelity :Does not have proof reading ability Error rate 1 in 2 X 104 bases Seems rare but can be recovered in cloning a single molecule Newer polymerases have high fidelity Problems with Taq


Slide 9:􀂄 Small amount of template 􀂄 In theory a single molecule 􀂄 Do not need to isolate sequence of interest 􀂄 DNA template need not be highly purified 􀂄 DNA is stable in absence of nucleases Templates for PCR


Slide 14:! Complete denaturation of the DNA template ! Optimal annealing temperature ! Optimal extension temperature ! Number of PCR cycles ! Final extension step PCR procedures: conditions for cycling


Slide 15:Contamination of the DNA must be prevented by: ! Separating the areas for DNA extraction and PCR ! Using sole-purpose laboratory equipment ! Autoclaving and aliquoting ! Adding a control reaction PCR procedures: precautions


Slide 16:Components: • Sterile deionised water • 10X PCR buffer • dNTP mix • Primer • Taq DNA polymerase • MgCl2 • Template DNA Considerations: • Template DNA • Primers • MgCl2 concentration • Taq polymerase • dNTPs PCR procedures: components


Slide 17:! Micropipettes ! Thermocycler ! Electrophoresis units ! Power supply units ! Photographic equipment PCR procedures: equipment


Good Primer’s Characteristic :Good Primer’s Characteristic A melting temperature (Tm) in the range of 52 0C to 65 0C Absence of dimerization capability Absence of significant hairpin formation (>3 bp) Lack of secondary priming sites Low specific binding at the 3' end (ie. lower GC content to avoid mispriming)


Slide 19:Uniqueness There shall be one and only one target site in the template DNA where the primer binds, which means the primer sequence shall be unique in the template DNA. There shall be no annealing site in possible contaminant sources, such as human, rat, mouse, etc. (BLAST search against corresponding genome) Primer candidate 1 5’-TGCTAAGTTG-3’ Primer candidate 2 5’-CAGTCAACTGCTAC-3’ TGCTAAGTTG CAGTCAACTGCTAC Template DNA 5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’ NOT UNIQUE! UNIQUE!


Length :Length Primer length has effects on uniqueness and melting/annealing temperature. The longer the primer, the more chance that it’s unique; the longer the primer, the higher melting/annealing temperature. The length of primer has to be at least 15 bases to ensure uniqueness. Usually primers of 17-28 bases long are used.


Melting Temperature :Melting Temperature Melting Temperature, Tm – the temperature at which half the DNA strands are single stranded and half are double-stranded.. Tm is characteristics of the DNA composition; Higher G+C content DNA has a higher Tm due to more H bonds. Calculation Shorter than 13: Tm= (wA+xT) * 2 + (yG+zC) * 4 Longer than 13: Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC) (Formulae are from http://www.basic.northwestern.edu/biotools/oligocalc.html)


Slide 22:Annealing Temperature Tanneal = Tm_primer – 4C Annealing Temperature, Tanneal – the temperature at which primers anneal to the template DNA. It can be calculated from Tm .


Internal Structure :Internal Structure If primers can anneal to themselves, or anneal to each other rather than anneal to the template, the PCR efficiency will be decreased dramatically. They shall be avoided. However, sometimes these 2 structures are harmless when the annealing temperature does not allow them to take form. For example, some dimers or hairpins form at 30 C while during PCR cycle, the lowest temperature only drops to 60 C.


Primer Pair Matching :Primer Pair Matching Primers work in pairs – forward primer and reverse primer. Since they are used in the same PCR reaction, it shall be ensured that the PCR condition is suitable for both of them. One critical feature is their annealing temperatures, which shall be compatible with each other. The maximum difference allowed is 3 C. The closer their Tanneal are, the better.


Summary ~ Primer Design Criteria :Summary ~ Primer Design Criteria Uniqueness: ensure correct priming site; Length: 17-28 bases.This range varies; Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible; Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming. Melting Tm between 55-80 C are preferred; Assure that primers at a set have annealing Tm within 2 – 3 C of each other. Minimize internal secondary structure: hairpins and dimmers shall be avoided.


PCR Primers :PCR Primers Identify gene sequence in mRNA sequence Select primers to use for RT and PCR 1 gtattaataa tgtcgacttc aggaactggt aagatgactc gcgcgcagcg tcgagctgcc 61 gctcgtagaa atcgtcggac cgctggggtc caaccagtaa ttgtcgaacc aatcgctgct 121 ggccaaggca aggccattaa agcgattgca ggatacagca tatcaaagtg ggaggcgtct 181 tcggacgcga ttacagcgaa agccaccaat gccatgagta tcactctgcc ccatgagctc 241 tcttctgaaa agaataagga gcttaaggtc ggcagagtgc tgctttggtt gggacttctt 301 cctagcgttg ctgggaggat taaggcttgt gttgctgaga aacaggcaca ggccgaggcc 361 gcttttcaag tagccttggc ggttgctgac tcctcgaaag aggtggtcgc ggccatgtat 421 acggacgcct ttcgaggggc gactctgggg gatttgctta atctccagat ttatctgtat 481 gcatctgaag cagtgcctgc taaggcggtc gttgtacatc tagaagttga gcacgtaagg 541 cctacgttcg atgacttctt caccccggtt tataggtagt gcccctgctc ggagagcccc 601 tgactgggtt aaagtcacag gccccttgtc tcaggtagag accctgtcca ggtaggacac 661 tttggctaag gttaaaagct tgttgaatca gtacaataac tgatagtcgt ggtttacacg 721 cagacctctt acaagagtgt ctaggtgcct ttgagagtta ctctttgctc tcttcggaag 781 aacccttagg ggttcgtgca tgggcttgca tagcaagtct tagaatgcgg gtaccgtaca 841 gtgttgaaaa acactgtaaa tctctaaaag agacca 1 gtattaataa tgtcgacttc aggaactggt aagatgactc gcgcgcagcg tcgagctgcc 61 gctcgtagaa atcgtcggac cgctggggtc caaccagtaa ttgtcgaacc aatcgctgct 121 ggccaaggca aggccattaa agcgattgca ggatacagca tatcaaagtg ggaggcgtct 181 tcggacgcga ttacagcgaa agccaccaat gccatgagta tcactctgcc ccatgagctc 241 tcttctgaaa agaataagga gcttaaggtc ggcagagtgc tgctttggtt gggacttctt 301 cctagcgttg ctgggaggat taaggcttgt gttgctgaga aacaggcaca ggccgaggcc 361 gcttttcaag tagccttggc ggttgctgac tcctcgaaag aggtggtcgc ggccatgtat 421 acggacgcct ttcgaggggc gactctgggg gatttgctta atctccagat ttatctgtat 481 gcatctgaag cagtgcctgc taaggcggtc gttgtacatc tagaagttga gcacgtaagg 541 cctacgttcg atgacttctt caccccggtt tataggtagt gcccctgctc ggagagcccc 601 tgactgggtt aaagtcacag gccccttgtc tcaggtagag accctgtcca ggtaggacac 661 tttggctaag gttaaaagct tgttgaatca gtacaataac tgatagtcgt ggtttacacg 721 cagacctctt acaagagtgt ctaggtgcct ttgagagtta ctctttgctc tcttcggaag 781 aacccttagg ggttcgtgca tgggcttgca tagcaagtct tagaatgcgg gtaccgtaca 841 gtgttgaaaa acactgtaaa tctctaaaag agacca


Computer-Aided Primer Design :Computer-Aided Primer Design Primer design is an art when done by human beings, and a far better done by machines. Some primer design programs we use: Oligo: Life Science Software, standalone application - GCG: Accelrys, ICBR maintains the server. Primer3: MIT, standalone / web application http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi BioTools: BioTools, Inc. ICBR distributes the license. Others: GeneFisher, Primer!, Web Primer, NBI oligo program, etc. Melting temperature calculation software: - BioMath: http://www.promega.com/biomath/calc11.htm


Task :Task Design a pair of primers for sequence “NM_203378” in NCBI GenBank, so that the coding sequence of human myoglobin will be amplified using PCR reaction. Between 156..620


Primer3 :Primer3 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi


Primer3 :Primer3


Primer3 :Primer3


Slide 32:www.fisheroligos.com  (sigma genosys) scale price/base     50 nmole   $0.75 200 nmole   $1.30 25 base oligo $18.75 www.fisheroligo.com  (operon) scale price/base     50 nmole   $0.50 200 nmole   $1.00 25 base oligo $12.50


Slide 33:Contamination Control: Air Control Aliquoting reagents Dedicated tools Radiating plastic ware Filter tips Controls Segregation (genomic from PCR, samples from nulls)


RT-PCR (Reverse transcriptase-PCR) :RT-PCR (Reverse transcriptase-PCR) For amplifying a defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into complementary DNA Amplification of the resulting DNA using polymerase chain reaction Extract RNA from virus/cells


Slide 35:Random Amplified Polymorphic DNA The target sequence's) (to be amplified) is unknown A primer with an arbitrary sequence


Single-Strand Conformational Polymorphism :Single-Strand Conformational Polymorphism Highly sensitive to DNA sequence: can detect single base changes Simple process but can be difficult to repeat 1. Amplify Target Sequence 2. Denature product with heat and formamide 3. Analyze on native (nondenaturing) polyacrylamide gel 4. Base sequence determines 3-dimensional conformation, and rate of migration


Denaturing Gradient Gel Electrophoresis :Denaturing Gradient Gel Electrophoresis 4. Denaturing gradient gels are difficult to produce: use perpendicular gradient to identify optimal conditions, move to CDGE: constant denaturant gel electrophoresis 1. Amplify Target Sequence 2. Run product on gel with denaturing gradient (parallel or perpendicular to direction gel runs) 3. Product begins denaturing at a certain point, depending on base sequence: greatly retards migration and allows discrimination of alleles based on small sequence differences


Slide 38:􀂄 Preimplantation diagnosis of genetic diseases 􀂄 Forensics 􀂄 Paternity testing Other application of PCR


In summary :In summary ! PCR is a simple technique to obtain many copies of specific DNA fragments ! Three steps are involved in PCR: denaturation, annealing and extension ! To ensure success, care should be taken both in preparing the reaction mixture and setting up the cycling conditions