ICS 2 Notes

1. Gel electrophoresis
a. Principles of DNA gel electrophoresis
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Used to separate charged molecules by their differential rates of movement in an electric field DNA molecules are separated based on their rate of migration through the gel matrix As the phosphate groups in the sugar-phosphate backbone are negatively charged, it migrates toward the positive electrodes The complex network of pores in the gel matrix acts as a molecular sieve to retard the movement of DNA molecules and separate them by size/length Shorter DNA fragments are less impeded by the pores than longer ones and move through the gel matrix more quickly, so that molecules of different length migrate as distinct bands. Thus, a complex mixture of linear DNA fragments is size-fractionated into discrete bands, each consisting of DNA molecules of the same length After electrophoresis, the gel is removed and stained with a DNA-binding dye such as methylene blue or ethidium bromide, this allows the separated DNA fragments to be visualized as a series of bands within the gel

b. Applications
To separate/purify DNA fragments according to size To determine the approximate molecular weight of the separated DNA fragments To identify/isolate individual fragments for further study (bands of interest can be excised from the gel) To check results of PCR ( to determine if PCR is successful)

2. Polymerase Chain Reaction (PCR)

a. Components of PCR
DNA sample (template) double stranded DNA sample which contains the nucleotide sequence of interest Thermostable DNA pol. TAQ polymerase from thermophillic bacterium (enzyme is stable at high temperatures and will not be denatured by repeated cycles of PCR) DNA oligonucleotide primers 2 sets of short single strander DNA oligonucleotide primers flank the region of interest and are complementary to the 3 end of both template strands. Free deoxyribonucleotides present in excess to synthesize new DNA strands PCR reaction buffer contains magnesium chloride as Mg2+ ions are cofactors for DNA pol. activity

b. The PCR cycle

The entire 3 step cycle is repeated 20-30 times. Newly-synthesized DNA strands will serve as templates for further DNA synthesis in subsequent cycles. Only the target sequence bracketed by the 2 primers is amplified because there are no primers attached anywhere else. Hence, 3 PCR cycles produce:

16 DNA strands 8 are exactly identical in length and sequence of the region of DNA to be amplified; 8 contain extra DNA downstream of the original sequence, which is replicated in the first few cycles 8 DNA molecules 2 are identical to the original template

c. Applications of PCR

Amplify a large number of copies of a DNA sequence from a small amount of original sample in a short time PCR specifically amplifies only the section of DNA between the 2 primers, hence it can also be used for purification; the PCR product will consist almost exclusively of the target DNA sequence

3. Nucleic Acid Hybridisation

Principles of NAH

a.

Process by which 2 complementary single-stranded nucleic acid chains base-pair and reform a double stranded hybrid

When kept for a prolonged period of time below 65 degrees, hydrogen bonds can re-establish and the 2 single strands readily anneal to re-form a double helix by DNA renaturation or hybridization Can occur between any 2 single-stranded nucleic acid chains: DNA/DNA, RNA/RNA, DNA/RNA, provided they have complementary nucleotide sequences Used to detect specific RNA and DNA nucleotide sequences using particular singlestranded nucleic acid probes (probes are single-stranded DNA or RNA and are labeled e.g. radioactive, fluorescent etc.)

b. Applications
To detect, characterize and quantify specific nucleotide sequences or genes in DNA/RNA molecules To localize particular genes of interest or families of related but non-identical genes To study gene expression and changes in gene expression profiles To screen libraries of cloned DNA or bacterial clones to identify clones carrying DNA insert of interest To compare nucleotide sequences between 2 DNA samples

c. Techniques utilizing NAH

1. Southern blotting Utilizes a DNA sample and thus allows the tester to detect the presence of a specific gene or DNA sequence 2. Northern blotting Utilizes an RNA sample and hence allows the study of gene expression 3. In situ hybridization 4. Colony hybridization

d. Process