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

2. Specification

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 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.

7. oC
INITIAL DENATURATION PRIMERS ANNEAL EXTENSION DENATURATION FINAL EXTENSION COOLING
50-65 oC
72 oC oC
72 oC
4-10 oC

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 from
The 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 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

11. The need for thermostability
Polymerase must withstand this temperature
But 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 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

14. PAST PAPER QUESTIONS

17. 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

18. 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

19. The uses of DNA probes

20. PAST PAPER QUESTIONS

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

22. 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

23. 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

24. 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.

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

26. 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

27. Microsatellite repeat sequences

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 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

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 gel
We 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?

34. PAST PAPER QUESTIONS