Module 1 Section 1.3 DNA Technology AS Biology Module 1 Section 1.3 DNA Technology

Specification

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.

Summary of the process The whole process can be summarised in a few simple steps: Firstly, the DNA template is heated to denature it into single strands Secondly, 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 present DNA polymerase (present in the reaction mix) begins to synthesise new DNA strands You now have two copies of the DNA you started with and the whole process repeats.

95-110 oC INITIAL DENATURATION PRIMERS ANNEAL EXTENSION DENATURATION FINAL EXTENSION COOLING 50-65 oC 72 oC 95-110 oC 72 oC 4-10 oC

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 from The primer acts as a double strand on the template molecule

Exercise Draw the primer for the second DNA template Draw the sequence of DNA produced following DNA synthesis

DNA Polymerase A thermostable enzyme It can withstand temperatures of 110 oC and still maintain its functionality The first PCR DNA polymerase to be used is called Taq and was isolated from Thermos aquaticus which was found to live in hot springs Scientists wondered how it replicated DNA and was able to live at such high temperatures and after studying it discovered its SPECIAL DNA polymerase

The need for thermostability Polymerase must withstand this temperature But polymerase only begins to work here

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

PCR uses PCR has a multitude of uses; Genetic Testing: to screen for and detect DNA mutations Tissue typing: before organ transplant to test for compatibility Genetic fingerprinting at crime scenes Paternity testing DNA sequencing DNA cloning Creating large volumes of DNA for other work Genetic mapping

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DNA Probes A DNA probe is a SHORT length of DNA with KNOWN nucleotide BASE SEQUENCE Either has a fluorescent or radioactively labelled end and so can be used as a marker

The uses of DNA probes The probe will base pair with any complementary nucleic acid strands As we know the probe sequence we can use it to probe and entire GENOME and find any sites of complementary DNA

The uses of DNA probes

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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 occur We can detect very subtle changes in DNA sequence between individuals with a high degree of accuracy, caused by genetic variation

Genetic Marker Sites Many diseases can be caused by single mutations in DNA sequence This 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

Types of Genetic Marker There are different types of genetic marker Restriction fragment length polymorphisms DNA 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

Types of Genetic Marker Single nucleotide polymorphism A 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.

Single nucleotide polymorphisms These can be detected by PCR One type of DNA One type of DNA One type of DNA

Types of Genetic Marker Microsatellite repeat sequences These 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 MRS 3 MRS 4 MRS 5 MRS 6 MRS Fluorescent probe

Microsatellite repeat sequences

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 scene The first step is to RESTRICTION DIGEST (using restriction endonucleases) chromosomal DNA, so as to chop it into smaller pieces Secondly, these fragments are separated according to size using DNA GEL ELECTROPHORESIS to produce a unique profile

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 gel We can locate specific DNA fragments this way, so if a particular RFLP is present, you can probe for it

EXAMPLE Which of these children is from the mother previous marriage? Which of these children is adopted?

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