Molecular Techniques

Question 1

Components of PCR

Answer 1

1. DNA template strand
2. DNA thermostable taq polymerase
3. deoxryibonucleoside triphosphate(dNTPs)
4. PCR primer
5. PCR reaction buffer

Question 2

Steps of PCR reaction

Answer 2

1. Reaction mixture is heated to 95 degree celcius for 30s to breaks the hydrogen bonds between the 2 strands of DNA template strand
2. reaction mixture cooled to 54degree celcius for 1 minute for annealing of RNA primers to complementary sequence on 3’ end of DNA template
3. reaction mixture heated to ~72 degree celcius for 2mins. RNA primer primes DNA template for synthesis of DNA strand using deoxyribonucleoside triphosphate. DNA synthesis catalysed by thermostable taq polymerase

Question 3

Limitations of PCR

Answer 3

• infidelity of PCR due to taq thermostable polymerase not having 3’ to 5’ exonuclease activity, cannot recognise incorrect base pairs and excise them
• extreme sensitivity of PCR, any contamination of reaction mixture with non-template nucleic acid causes the non-template nucleic acid to be amplified
• can only amplify DNA template length of a few thousand kb
• need to construct DNA primers means prior sequence information is needed

Question 4

Mechanisms of gel electrophorses

Answer 4

• agarose gel is a gel matrix that acts as a “molecular sieve” that impedes movement of DNA fragments to separate DNA fragments by length or size
• direct current is applied to gel matrix, negatively charged sugar-phosphate backbone causes DNA fragment to move towards positively charge electrode(anode)
• shorter fragments is less impeded by gel matrix annd moves quicker through gel maxtrix, longer fragments more impeded by gel matrix and moves faster through gel matrix
• different length migrate as distinct bands
• DNA is size-fractioned

Question 5

Definition of nucleic acid hybridisation

Answer 5

process by which 2 complementary, single stranded nucleic acids base pairs to reform a double-stranded hybrid

Question 6

Process of nucleic acid hybridisation

Answer 6

• denaturation of DNA double helix into 2 single-stranded nucleic acids via prolonged exposure to temperature above 100 degree celcius, pH≥13, low salt concentrations
• prolonged exporsure to temperature of 65 degree celcius and below allows for hydrogen bonds between complementary base pairs to reform, allowing the 2 single-stranded nuclei acid to readily anneal to each other and reform DNA double helix

Question 7

Application of nucleic acid hybridisation

Answer 7

• identify specific base sequence of RNA and DNA using specific single-stranded nucleic acid probe

Question 8

Probe molecule

Answer 8

• single-stranded nucleic acid
• labelled, carry radioactive, flourescent or chemical markers to facilitate detection
• cloned from genomic or cDNA molecules

Question 9

Advtantages of nucleic acid hybridisation

Answer 9

1. Sensitive: complementary sequences at low concentrations can be detected
2. Selective: nucleic acid probes only hybridises to nucleic acid molecules that contain all or part of the complementary sequence

Question 10

Applications of nucleic acid hybridisation

Answer 10

1. locate, characterise, quantify specific nucleotide sequences in RNA/DNA molecules
2. locate particular genes of interest or families of related but non-identical genes in cells, tissues and organisms
3. study gene expression and changes in gene expression profile
4. screen libraries of cloned DNA to identity gene insert or interest in clones/colonies
5. compare base/nucleotide sequence of two DNA/RNA molecules in phylogeny studies

Question 11

Principal of southern blotting