Lab Techniques

Question 1

Describe the steps in PCR.

Answer 1

1) Denaturation @ 95C
2) Annealing of primers @ 55C
3) Elongation with heat-stable DNA polymerase @ 72C

See First Aid page 48

Question 2

Southern vs. Northern vs. Western vs. Southwestern Blots

Answer 2

Southern - DNA; sample enzymatically cleaved and separated onto gel, when transferred onto filter; filter exposed to radiolabeled DNA probe

Northern - RNA; similar to southern

Western - Protein; similar to southern but use labeled antibody to detect relevant protein

Southwestern - DNA binding proteins (i.e. transcription factors) identified with labeled oligonucleotide probes

See First Aid page 49

Question 3

Microarrays

Answer 3

Thousands of nucleic acid sequences arranged on glass or silicon. DNA or RNA probes are hybridized to chip and scanner detects relative amounts of complementary binding.

Able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs)

See First Aid page 50

Question 4

ELISA (enzyme-linked immunosorbent assay)

Answer 4

Used to detect specific antigen (e.g. HBsAg) or antibody (e.g. anti-HBs) in patient’s blood sample.

Detection with antibody linked to enzyme. Added substrate then reacts with enzyme to produce detectable signal.

Can have high sensitivity and specificity but less specific than Western.

See First Aid, page 52

Question 5

Cre-lox system

Answer 5

Inducibly manipulate genes at specific developmental points (eg to study genes whose deletions cause embryonic death).

See First Aid, page 52

Question 6

RNA interference

Answer 6

dsRNA synthesized to be complementary to mRNA sequence of interest. It is then transfected into human cells, where it separates and promotes degradation of target mRNA, thereby “knocking down” (NOT KNOCKING OUT) gene expression.

See First Aid, page 52