Mol Methods

Question 1

Which technique is required to carry out a RFLP assay?

Answer 1

Southern blot

Question 2

southern blots use what type of probes?

Answer 2

nucleic acid probes.

Question 3

PCR analysis

Answer 3

• Taq polymerase to copy DNA b/w primers.
• Remelt, repeat reaction multiple times (amplification)
• It is highly specific

Question 4

DNA Sequencing

Answer 4

• Info on specific nucleotides

- Takes a long time, expensive

Question 5

RNA Methods: Northern Blot

Answer 5

• Denature RNA to make it linear
• Nucleic acid probe complementary to RNA
• Unlike southern blot, you can QUANTIFY the amount (of RNA though, not DNA)

Question 6

RNA Methods: RT- PCR

Answer 6

• Converts mRNA into DS DNA using reverse transcriptase and a primer
• Exonuclease degrades old mRNA
• Run DNA in PCR assay.

Question 7

RNA Methods: Microarray

Answer 7

• Labeled RNA hybridized to unlabeled probe (opposite of northern blot)
• mRNA with probe then hybridized on a spot-plate.
• Idea of HOW MUCH mRNA is present.
• Large-scale analysis

Question 8

Southern vs. Western vs. Northern: Differences in what they do?

Answer 8

• Western blot uses antibody probe, whereas southern and northern use nucleic acid probes
• Northern and Western use denaturing gels, whereas southern does not
• Southern (RFLP analysis) used for presence of DNA sequence, NOT amount of DNA
• Northern can quantify amount of RNA, and has to make RNA linear (denaturing)