ICS 2 Notes

8/2/2019 ICS 2 Notes

1/6

ICS 2 Notes

1. Gel electrophoresis

a. Principles of DNA gel electrophoresis

- 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 negativelycharged, 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)


2/6

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 forDNA pol. activity


3/6

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:


4/6


5/6

- 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 single-

stranded 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


6/6

d. Process

ICS 2 Notes

Documents

Transcript of ICS 2 Notes