Polymerase Chain Reaction(PCR) The most important discovery in biology since thediscovery of the structure of DNA! Years ago, when doing analysis of DNA, if DNAwas lost or contaminated it could take months tomake enough to allow a re-analysis! The discovery of the PCR as a technique toquickly and accurately create more copies of apiece of DNA of interest was revolutionary.
Summary of the process The whole process can be summarised in a fewsimple steps:1. Firstly, the DNA template is heated to denature itinto single strands2. Secondly, the temperature is reduced and primersSPECIFIC to each of the two denatured DNAstrands are added. The primers anneal to the DNAstrands due to complementary bases being present3. DNA polymerase (present in the reaction mix)begins to synthesise new DNA strands4. You now have two copies of the DNA you startedwith and the whole process repeats.
Primers and how they function To begin synthesis ofnew DNA, DNApolymerases need aregion of doublestranded DNA to actas a template that theycan bind to so as tobegin transcriptionfrom The primer acts as adouble strand on the
Exercise1. Draw the primer forthe second DNAtemplate2. Draw the sequenceof DNA producedfollowing DNAsynthesis
DNA Polymerase A thermostable enzyme It can withstand temperatures of 110 oC and stillmaintain its functionality The first PCR DNA polymerase to be used iscalled Taq and was isolated from Thermosaquaticus which was found to live in hot springs Scientists wondered how it replicated DNA andwas able to live at such high temperatures andafter studying it discovered its SPECIAL DNApolymerase
The need for thermostabilityButpolymeraseonly beginsto workherePolymerasemustwithstandthistemperature
Copies of DNA you can make After 30 cycles(which will takeabout 2 hours tocarry out) you willhave 536 MILLIONcopies of yourstarting template
PCR uses PCR has a multitude of uses; Genetic Testing: to screen for and detect DNAmutations Tissue typing: before organ transplant to test forcompatibility Genetic fingerprinting at crime scenes Paternity testing DNA sequencing DNA cloning Creating large volumes of DNA for other work Genetic mapping
DNA Probes A DNA probe is a SHORT length of DNA withKNOWN nucleotide BASE SEQUENCE Either has a or labelledend and so can be used as a marker
The uses of DNA probes The probe will base pair with any complementarynucleic acid strands As we know the probe sequence we can use it toprobe and entire GENOME and find any sites ofcomplementary DNA
Detecting minor differences inDNA Genetic variation can be caused by either naturalchanges in DNA sequence over time, or else bymutations that spontaneously occur We can detect very subtle changes in DNAsequence between individuals with a high degreeof accuracy, caused by genetic variation
Genetic Marker Sites Many diseases can be caused by singlemutations in DNA sequence This one change can lead to a change in aminoacid in a protein or no protein at all! Sites that regularly show differences are referredto as genetic “marker” sites
Types of Genetic Marker There are different types of genetic marker Restriction fragment length polymorphisms DNA molecules are cut by restriction nucleases. Ifthere is a mutation that has occurred at the site ofone of the restriction sites then we will get adifferent length DNA fragment following PCR
Types of Genetic Marker Single nucleotide polymorphism A DNA sequence variation occurring when aSINGLE nucleotide in the genome differs betweenmembers of a biological species or pairedchromosomes in a human. IF two genes have aSNP then they are referred to as alleles.
Single nucleotide polymorphisms These can be detected by PCROne type of DNAOne type of DNAOne type of DNA
Types of Genetic Marker Microsatellite repeat sequences These are di-,tri- or tetra nucleotide tandemrepeats in DNA sequences. The number varieswithin populations and within a persons alleles.You can detect them by PCR as you will getbigger and bigger products the more repeats youhave.2MRS3MRS4Fluorescent probe
Genetic Fingerprinting Genetic fingerprinting is a powerful technique thatcan allow identification of a person at a scene justby comparing DNA of the person with some DNAfound at the scene The first step is to RESTRICTION DIGEST (usingrestriction endonucleases) chromosomal DNA, soas to chop it into smaller pieces Secondly, these fragments are separatedaccording to size using DNA GELELECTROPHORESIS to produce a unique profile
Probes and Fingerprinting If you want to increase the reliability of the resultyou can use DNA PROBES to further identifyspecific bands within the gel We can locate specific DNA fragments this way,so if a particular RFLP is present, you can probefor it
EXAMPLE Which of these children is from the motherprevious marriage? Which of these children is adopted?