Applied molecular biology

Question 1

steps for PCR

Answer 1

Denature (separate the strands) DNA (heat to 92-95C)
Anneal primers to DNA (cool to 50-65C)
Synthesize new DNA strands (heat to 68-72C)

Question 2

Nested PCR

Answer 2

More specific than PCR because two sets of primers need to bind to target

Question 3

qt PCR

Answer 3

Fluorescence caused by amplification

Question 4

rtPCR

Answer 4

Copy RNA into DNA (cDNA) using reverse transcriptase
Use cDNA to perform PCR
Detect RNA viruses

Question 5

advantages of PCr

Answer 5

Sensitive- detect 1 molecule
Accurate- distinguish between closely related sequences
Fast- hours

Question 6

hybridization

Answer 6

Binding of labeled probes to RNA or DNA

Question 7

Southern Blot

Answer 7

Southern Blots (detection of DNA fragments) were described by Southern in 1975
DNA fragments are separated by electrophoresis
DNA is denatured and transferred to a nitrocellulose or nylon membrane
Membranes are baked to link the DNA to the filter
Small pieces of labeled DNA (probes) are hybridized to the filter. The sequence of the probe determines which DNA fragments are detected

Question 8

Northern Blot

Answer 8

Northern blots are a modification of Southern blots and detect specific RNA fragments

Question 9

in situ hybridization

Answer 9

Used to localize RNAs (or pathogen DNAs) within tissues.
Fix tissue sample and unmask RNAs
Hybridize radioactive or enzymatic linked probe to tissue sample
Detect probe

Question 10

Gene chip

Answer 10

Label two sets of RNAs (i.e. infected versus uninfected cells) with dyes that fluoresce red and green
Hybridize RNAs to the DNAs on the “chip”
Measure dye intensity on each spot and analyze using a computer