3 Polymerase Chain Reaction (PCR) The most important discovery in biology since the discovery of the structure of DNA!Years ago, when doing analysis of DNA, if DNA was lost or contaminated it could take months to make enough to allow a re-analysis!The discovery of the PCR as a technique to quickly and accurately create more copies of a piece of DNA of interest was revolutionary.
4 Summary of the processThe whole process can be summarised in a few simple steps:Firstly, the DNA template is heated to denature it into single strandsSecondly, the temperature is reduced and primers SPECIFIC to each of the two denatured DNA strands are added. The primers anneal to the DNA strands due to complementary bases being presentDNA polymerase (present in the reaction mix) begins to synthesise new DNA strandsYou now have two copies of the DNA you started with and the whole process repeats.
8 Primers and how they function To begin synthesis of new DNA, DNA polymerases need a region of double stranded DNA to act as a template that they can bind to so as to begin transcription fromThe primer acts as a double strand on the template molecule
9 Exercise Draw the primer for the second DNA template Draw the sequence of DNA produced following DNA synthesis
10 DNA Polymerase A thermostable enzyme It can withstand temperatures of 110 oC and still maintain its functionalityThe first PCR DNA polymerase to be used is called Taq and was isolated from Thermos aquaticus which was found to live in hot springsScientists wondered how it replicated DNA and was able to live at such high temperatures and after studying it discovered its SPECIAL DNA polymerase
11 The need for thermostability Polymerase must withstand this temperatureBut polymerase only begins to work here
12 Copies of DNA you can make After 30 cycles (which will take about 2 hours to carry out) you will have 536 MILLION copies of your starting template
13 PCR uses PCR has a multitude of uses; Genetic Testing: to screen for and detect DNA mutationsTissue typing: before organ transplant to test for compatibilityGenetic fingerprinting at crime scenesPaternity testingDNA sequencingDNA cloningCreating large volumes of DNA for other workGenetic mapping
17 DNA ProbesA DNA probe is a SHORT length of DNA with KNOWN nucleotide BASE SEQUENCEEither has a fluorescent or radioactively labelled end and so can be used as a marker
18 The uses of DNA probesThe probe will base pair with any complementary nucleic acid strandsAs we know the probe sequence we can use it to probe and entire GENOME and find any sites of complementary DNA
21 Detecting minor differences in DNA Genetic variation can be caused by either natural changes in DNA sequence over time, or else by mutations that spontaneously occurWe can detect very subtle changes in DNA sequence between individuals with a high degree of accuracy, caused by genetic variation
22 Genetic Marker SitesMany diseases can be caused by single mutations in DNA sequenceThis one change can lead to a change in amino acid in a protein or no protein at all!Sites that regularly show differences are referred to as genetic “marker” sites
23 Types of Genetic Marker There are different types of genetic markerRestriction fragment length polymorphismsDNA molecules are cut by restriction nucleases. If there is a mutation that has occurred at the site of one of the restriction sites then we will get a different length DNA fragment following PCR
24 Types of Genetic Marker Single nucleotide polymorphismA DNA sequence variation occurring when a SINGLE nucleotide in the genome differs between members of a biological species or paired chromosomes in a human. IF two genes have a SNP then they are referred to as alleles.
25 Single nucleotide polymorphisms These can be detected by PCROne type of DNAOne type of DNAOne type of DNA
26 Types of Genetic Marker Microsatellite repeat sequencesThese are di-,tri- or tetra nucleotide tandem repeats in DNA sequences. The number varies within populations and within a persons alleles. You can detect them by PCR as you will get bigger and bigger products the more repeats you have.2 MRS3 MRS4 MRS5 MRS6 MRSFluorescent probe
30 Genetic Fingerprinting Genetic fingerprinting is a powerful technique that can allow identification of a person at a scene just by comparing DNA of the person with some DNA found at the sceneThe first step is to RESTRICTION DIGEST (using restriction endonucleases) chromosomal DNA, so as to chop it into smaller piecesSecondly, these fragments are separated according to size using DNA GEL ELECTROPHORESIS to produce a unique profile
32 Probes and Fingerprinting If you want to increase the reliability of the result you can use DNA PROBES to further identify specific bands within the gelWe can locate specific DNA fragments this way, so if a particular RFLP is present, you can probe for it
33 EXAMPLE Which of these children is from the mother previous marriage? Which of these children is adopted?