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Introduction to the Polymerase Chain Reaction Amplification (PCR

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The Invention of PCR
• Invented by Kary Mullis in 1983 • Awarded Nobel Prize for Chemistry in 1993
You can read his Nobel lecture here: http://nobelprize.org/nobel_prizes/chemistry/laureates/199 3/mullis-lecture.html

What is PCR?
• Polymerase Chain Reaction = repeated, sequencespecific amplification of nucleic acid (usually DNA). It’s quick (can only take a few hours – result). It’s sequence-specific – the ends of the amplified piece of nucleic acid are define by the design of the primers. By molecular biology standards it’s quite cheap.

Why is it the ‘in’ technique & why is it so useful? • •

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• 2 . Repeats the equivalent of 30 – 40 rounds of semi. Uses DNA polymerases to add deoxyribonucleotides to a growing DNA strand complementary to the target sequence • DNA replication always proceeds in a 5’ to 3’ direction.. • HOW DO WE MIMIC DNA REPLICATION IN VITRO? • • Supply all components and incubate at 37°C ? – NO Generally need to know some details of the DNA sequence of interest . Different STRs found at different loci.e. Individuals can be homozygous or heterozygous for a particular STR • Each person has a particular set of STR alleles of different loci – analyse enough and this becomes a ‘unique’ profile within a population – used to link individuals to biological material found at crimes scenes….Why? Allows the design of oligonuleotide primers which are needed for the DNA polymerase to initiate polymerisation – i.conservative DNA replication in a few hours. •PCR is used to specifically amplify STR loci therefore identify allele profiles… How Does It Work? • • PCR mimics cellular DNA replication in vitro (inglass). DNA synthesis.• PCR is a Key technique in the Forensic analysis of DNA • Genetic profiling in humans based on the analysis of short tandem repeat sequences – STR’s ATCG¦ATCG¦ATCG¦ATCG¦ATCG¦ATCG¦ATCG¦ATCG = 8 repeats ATCG¦ATCG¦ATCG¦ATCG¦ATCG¦ATCG = 6 repeats Allele – 8 and Allele – 6 Position of an STR on a chromosome – locus.

the hydrogen bonds are so strong that the primer stays attached Extension at around 72°C : The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3'. all enzymatic reactions stop (for example the extension from a previous cycle). the double strand melts open to single stranded DNA. which can heat and cool the reaction tubes in a very short time. Typically: 94°C for 5 min – denature the target 94°C for 1 min) 55-60°C for 1 min) 72°C for 1-5 min) Plus: 72°C for 10 min to ensure amplicons (target amplified) are complete *annealing temp is varied 35-70 °C until optimised for product yield. Denaturation at around 94°C : During the denaturation. bases are added complementary to the template) Choice of cycling parameters: determined empirically. which are repeated for 20 to 40 cycles. This is done on an automated Thermo Cycler. Annealing at around 54°C : Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template. reading the template from 3' to 5' side.The different steps of PCR The cycling reactions : There are three major steps in a PCR. x35 denaturation primer annealing* primer extension 3 .

The temperature profile of a PCR cycle is controlled by the thermal cycler program which results in a near exponential increase in PCR product accumulation for about the first 30 cycles. They are normally sequence specific and therefore target specific. For a PCR reaction we need to supply: • • • • • • • • Template DNA (Target) Downstream Oligonucleotide Primer Upstream Oligonucleotide Primer Taq DNA Polymerase Reaction Buffer (x10 NH4 buffer) Cofactor . • • 4 . The primers must have their 3’OH ends pointing towards each other such that they flank the sequence to be amplified – primers are ‘used up’ in the process and become part of the product.MgCl2 Nuclease Free Water Nulease Free Light Mineral Oil (sometimes) • Deoyribonucleotide triphosphates = dNTPs Oligonucleotide Primer Design Criteria: • Require a primer for each end of the two strands that will define the ‘ends’ of the molecule synthesised.

temperature changes are used to: • Denature the target – make the double stranded DNA molecule single-stranded Anneal (stick) the primers to their complementary sequences Extend the primers by enzymatic addition of dNTPs to the end of the primers (3’OH) to ‘copy’ the strands of the duplex • • • All things being optimal.g. molecules after n cycles) 5 . 30 rounds of the cycle You should end up with – 1 073 741 824 = 230 molecules of amplicon X = 2n (where n=no.Primer Considerations: • • • • Ideally should only bind to the sequence of interest… Should be ~ 18-40 bases long… Should have a ‘high’ G+C content… Primer needs to be exact match with target – DNA polymerase needs a fully matched primer template sequence to begin polymerisation. it results in a roughly geometric increase in PCR amplicon (the bit you are amplifying) This assumes that you: • • • • Start with a single target molecule Carry out e. To mimic cellular DNA replication.

024 Expressed as an Exponent 20 21 22 ect 210 This assumes that the following conditions hold true: • • • • • • Start with 1 target molecule An excess of primers & dntps are available Over time buffer conditions do not change – pH etc. The exponential phase lasts for about 30 cycles under standard reactions conditions. Product Molecules 1 (initial target) 2 4 etc 1. Each step of the reaction has the same buffer & pH requirements 100% priming etc occurs on every molecule at each step Product inhibition does not occur occurs at ~1012 molecules / 100 µl of reaction PCR target molecules accumulate as a function of cycle number. The plateau phase results from limiting amounts of enzyme and reduced enzyme activity 6 .Exponential Amplification: Cycle Number 0 (start) 1 2 etc 10 No.

Teflon Coated Copper. ethylene glycol – better uniform heating of reaction vessels • Type and distribution of heating mechanism – usually an electrically heated coil • Type of cooling mechanisms – water-cooled. microtitre plates… • Individual rows of receptacles can be heated – can try lots of reaction temperatures in one ‘run’ 7 .How are these quick sequential changes of temperature achieved? • Heats & Cools quickly: • Block material – Silver.5ml..g. • Use of liquid medium – e. fan… • Programmable • Versatile reaction vessel capacity – 0.