PCR and gel electrophoresis

The polymerase chain reaction (PCR) and gel electrophoresis both work with molecules. Both these procedures are needed for forensic science.







Polymerase chain reaction (PCR) - rapid production of a large number of copies of a particular DNA fragment

1. DNA is denatured at 95 degrees Celcius --> separate DNA strands to expose bases
2. attach primers to ends of single-stranded DNA at 65 degrees Celcius
3. elongation: DNA polymerase builds new strands of DNA against exposed ones at 72 degrees Celcius

        * Taq polymerase - 1st heat-stable polymerase used in PCR

             - not destroyed in denaturation: no need to be replaced after each cycle

             - high optimum temperature to maximize efficiency 

                  --> temperature doesn't need to be dropped for annealing process

Gel electrophoresis - to separate different molecules; analysis of proteins and DNA)

- place mixture of molecules into wells cut in agarose gel --> apply electric field

- factors affecting movement speed of molecules:

• net charge of molecules
• size of molecule
• composition of gel: size of 'pores ' within gel

Electrophoresis of proteins 
- separates polypeptides produced by different alleles of many genes

• charge on protein is dependent on the ionisation of the R groups on the amino acid residues
• charge depends on pH --> use buffer solution to keep constant pH
• proteins are usually negatively charged
• polypeptides separate due to different net charges

Electrophoresis of DNA

• a region of DNA is chosen
• DNA is extracted
• DNA is chopped into pieces using restriction endonucleases
• fragments are transferred to absorbent paper --> placed onto gel --> heated to separate DNA strands
• "probes" (short sequences of single-stranded DNA) are added. They contain radioactive P isotopes --> X-ray results --> darken film, the separated fragments become visible


 

19.1 Principles of genetic technology  Genetic engineering involves the manipulation of naturally occurring processes and enzymes.  Genome sequencing gives information about the location of genes and provides evidence for the evolutionary links between organisms. a) define the term recombinant DNA  b) explain that genetic engineering involves the extraction of genes from one organism, or the synthesis of genes, in order to place them in another organism (of the same or another species) such that the receiving organism expresses the gene product  c) describe the principles of the polymerase chain reaction (PCR) to clone and amplify DNA (the role of Taq polymerase should be emphasised)  d) describe and explain how gel electrophoresis is used to analyse proteins and nucleic acids, and to distinguish between the alleles of a gene (limited to the separation of polypeptides and the separation of DNA fragments cut with restriction endonucleases)  e) describe the properties of plasmids that allow them to be used in gene cloning  f) explain why promoters and other control sequences may have to be transferred as well as the desired gene  g) explain the use of genes for fluorescent or easily stained substances as markers in gene technology  h) explain the roles of restriction endonucleases, reverse transcriptase and ligases in genetic engineering  i) explain, in outline, how microarrays are used in the analysis of genomes and in detecting mRNA in studies of gene expression