{"id":671,"date":"2014-04-30T12:45:41","date_gmt":"2014-04-30T12:45:41","guid":{"rendered":"http:\/\/hyndland-sec-glasgow.blogs.rm.com\/CfE-Higher-Unit-1\/?page_id=14"},"modified":"2014-04-30T12:45:41","modified_gmt":"2014-04-30T12:45:41","slug":"polymerase-chain-reaction","status":"publish","type":"page","link":"https:\/\/blogs.glowscotland.org.uk\/gc\/hyndsecbiohu1\/polymerase-chain-reaction\/","title":{"rendered":"Polymerase Chain Reaction"},"content":{"rendered":"
(b) Polymerase chain reaction (PCR) amplification of DNA using complementary primers for specific target sequences. DNA heated to separate strands then cooled for primer binding. Heat tolerant DNA polymerase then replicates the region of DNA. Repeated cycles of heating and cooling amplify this region of DNA. Practical applications of PCR.<\/span><\/h5>\n\n\n\n
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Homework Sheets 1_3<\/a>\/ 1_3A<\/a><\/p>\n

Abridged from : http:\/\/www.nature.com\/scitable\/definition\/polymerase-chain-reaction-pcr-110<\/a><\/h6>\n

Polymerase chain reaction, or PCR, is a laboratory technique used to make multiple copies of a segment of DNA.\u00a0 PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules.\u00a0 The following steps are used. Setting up the tubes:<\/p>\n

1.\u00a0 two short DNA sequences called primers<\/a> are designed to bind to the start and end of the DNA target.
\n2.\u00a0to perform PCR, the DNA template that contains the target is added to a tube that contains primers, free nucleotides, and an enzyme called DNA polymerase, and the mixture is placed in a PCR machine.
\n3.\u00a0 the PCR machine increases and decreases the temperature of the sample in automatic, programmed steps.<\/p>\n\n\n\n
\"DNAi\"<\/a><\/p>\n

Interactive animation of PCR<\/p>\n<\/td>\n

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The temperature changes which form the steps in the PCR process are necessary as a result of the the way in which DNA polymerase works.<\/p>\n

1.\u00a0 920<\/sup>-96o<\/sup>–C – DNA polymerase only works on single stranded template DNA, so the relatively high temperature is necessary to break the hydrogen bonds and create single stranded template DNA. (this is also why we use Taq polymerase a high temperature version of DNA polymerase).<\/p>\n

2.\u00a0 Cooling to aorund 55o<\/sup>-C – the primers will not bind\u00a0to the template strand at high temperatures (hydrogen bonds break at these temperatures), so cooling is required to allow the primers to bind (anneal).<\/p>\n

3.\u00a0 Heating again to around 72o<\/sup>-C – this allows the Taq polymerase to work efficiently to produce\u00a0replicate the DNA strand of the target DNA.<\/p>\n

Carrying out a cycle:
\na)\u00a0 Initially, the mixture is heated to between 92o<\/sup>–97o<\/sup>C to denature, or separate, the double-stranded DNA template into single strands.
\nb)\u00a0 The mixture is then cooled so that the primers anneal, or bind, to the DNA template.
\nc)\u00a0 At this point, the DNA polymerase begins to synthesize new strands of DNA starting from the primers.<\/p>\n

\"EssGen_PCR2-cycle3_MID_0\"<\/a><\/p>\n

Following synthesis and at the end of the first cycle, each double-stranded DNA molecule consists of one new and one old DNA strand. PCR then continues with additional cycles that repeat the aforementioned steps. Every cycle doubles the\u00a0number of copies\u00a0of target DNA present, because the newly synthesised DNA segments serve as templates in later cycles, giving exponential amplification up to\u00a0millions of times.<\/p>\n

Primers & specificty of PCR<\/h2>\n

The key to the specificity of PCR is the primers.\u00a0 The primers are\u00a0designed to locate a DNA sequence on either side of the target gene.\u00a0\u00a0The primer is exactly complementary to a short (20-30 bases) sequence of DNA on either side of the target and so will bind only to that sequence.\u00a0\u00a0 In this way the primer can be considered to find the “needle in the haystack”.\u00a0 PCR then produces millions or billions of copies of this single sequence – producing “haystacks from needles”.<\/p>\n

A short primer will be less specific.\u00a0 The chance of any base appearing at any position in the genome is one in 4 (4 bases).\u00a0 Therefore if your primer is only 2 bases long there is a 1 in 16 chance of encountering that random 2 base sequence in the genome.\u00a0 Given that the genome is 3 billion or so bases long – you will likely encounter a 2 base sequence\u00a0very often – so that primer will not provide specificity.\u00a0 A 20 base primer gives a 1 in 1,099,511,627,776, that’s a 1 in a trillion chance of encountering the same sequence randomly in the genome.<\/p>\n

Applications of PCR<\/h2>\n

PCR is simply a technique to amplify DNA, but if appropriate regions of DNA are amplified, PCR becomes a very powerful application in a variety of contexts.\u00a0 It is particularly useful if there are only small amounts of DNA available.
\nInfection diagnosis <\/strong>: PCR can be used to amplify specific parts of a pathogen’s genome to help identify the cause of an infectious disease
\nPaternity\/ Family tree <\/strong>: PCR is able to be used in providing confirmation of family relationships. It relies on the identification of differences in the repeated sequences present in geneomes (the so-called genetic fingerprint). These are inherited from both parents.
\nForensics<\/strong> : PCR\u00a0enables the analysis of miniscule amounts of DNA and consequent identification of the person responsible for leaving that DNA at crime scenes. It has also been used to exonorate serving prisoners and can be important in reversing miscarriages of justice.
\nAnthropology<\/strong> : PCR can be used to analyse the small amounts of DNA found in historic artefacts helping to identify relationships between individuals\u00a0and sometimes uniquely identify individuals who died hundreds or thousands of years ago.
\nPhyolgeny<\/strong> : PCR can help in the analysis evolutionary relationships between different species.<\/p>\n","protected":false},"excerpt":{"rendered":"

(b) Polymerase chain reaction (PCR) amplification of DNA using complementary primers for specific target sequences. DNA heated to separate strands then cooled for primer binding. Heat tolerant DNA polymerase then replicates the region of DNA. Repeated cycles of heating and cooling amplify this region of DNA. Practical applications of PCR. Homework Sheets 1_3\/ 1_3A Abridged … Continue reading “Polymerase Chain Reaction”<\/span><\/a><\/p>\n","protected":false},"author":2454,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"open","ping_status":"open","template":"","meta":{"footnotes":""},"class_list":["post-671","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/blogs.glowscotland.org.uk\/gc\/hyndsecbiohu1\/wp-json\/wp\/v2\/pages\/671","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/blogs.glowscotland.org.uk\/gc\/hyndsecbiohu1\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/blogs.glowscotland.org.uk\/gc\/hyndsecbiohu1\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/blogs.glowscotland.org.uk\/gc\/hyndsecbiohu1\/wp-json\/wp\/v2\/users\/2454"}],"replies":[{"embeddable":true,"href":"https:\/\/blogs.glowscotland.org.uk\/gc\/hyndsecbiohu1\/wp-json\/wp\/v2\/comments?post=671"}],"version-history":[{"count":0,"href":"https:\/\/blogs.glowscotland.org.uk\/gc\/hyndsecbiohu1\/wp-json\/wp\/v2\/pages\/671\/revisions"}],"wp:attachment":[{"href":"https:\/\/blogs.glowscotland.org.uk\/gc\/hyndsecbiohu1\/wp-json\/wp\/v2\/media?parent=671"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}