Module 1 Section 1.3 DNA Technology AS Biology. Specification.

AS Biology

Module 1 Section 1.3DNA TechnologyAS BiologySpecification

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 processThe 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.

INITIAL DENATURATION

PRIMERS ANNEALEXTENSIONDENATURATION

FINAL EXTENSION

COOLING95-110 oC72 oC95-110 oC72 oC4-10 oC50-65 oCPrimers and how they functionTo 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

ExerciseDraw the primer for the second DNA template

Draw the sequence of DNA produced following DNA synthesis

DNA PolymeraseA thermostable enzymeIt 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 polymeraseThe need for thermostabilityBut polymerase only begins to work here

Polymerase must withstand this temperatureCopies of DNA you can makeAfter 30 cycles (which will take about 2 hours to carry out) you will have 536 MILLION copies of your starting template

PCR usesPCR 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 mappingPAST PAPER QUESTIONS

DNA ProbesA 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 probesThe 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

PAST PAPER QUESTIONS

Detecting minor differences in DNAGenetic 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 SitesMany 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 sitesTypes of Genetic MarkerThere are different types of genetic marker

Restriction 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

Types of Genetic MarkerSingle 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.

Single nucleotide polymorphismsThese can be detected by PCR

One type of DNAOne type of DNAOne type of DNATypes of Genetic MarkerMicrosatellite 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 probeMicrosatellite repeat sequences

Genetic FingerprintingGenetic 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 FingerprintingIf 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

EXAMPLEWhich of these children is from the mother previous marriage?

Which of these children is adopted?

PAST PAPER QUESTIONS