BC 22 Molecular Bio in Med

Question 1

Restriction enzymes

Answer 1

bacterial enzymes
in nature used to destroy bacteriophages

• cuts dsDNA
• sequence specific
• orientation specific

Question 2

gels

Answer 2

agarose: larger pore size, few hundred base pair
polyacrylamide: smaller, one bp diff

Question 3

RE in disease mutation verification

Answer 3

cute the alle, normally would cut into two pieces, only cuts into one…diseased

Question 4

Sanger

Answer 4

enzymatic dideoxyribonucleotides
-missing 3' hydroxyl
-primer fluorescently labelled
-four different reaction with five different components
A, C, T, G reactions

-one of r at 1/100th amount of corresponding dNTP allows chain to extend and only occasionally terminate

RUN on polyacrylamide gel, each letter will have a lane and different lengths
-read from bottom to top

Question 5

Automated Sanger

Answer 5

fluorescent DNA

• all reactions in one tube and separated in one lane
• each lane displays the sequence obtained from a separate DNA sample and primer

-run by imaging using laser and computer for peaks that display correct base

Question 6

PCR

Answer 6

dsDNA
2 primers
excess dNTPs
Taq Polymerase

denaturation, annealing, and elongation

Nth cycle 2^n molecules

Question 7

RT PCR

Answer 7

rna-cdna-qPCR

Question 8

blotting

Answer 8

all radioactive or flourescent
southern: copy number of a specific gene
northern: measure size and amounts of mRNA
Western: enzyme linked antibody, measure amounts of proteins

need correct base pairing of complementary strands and probe, denature at high temp, hybridize at lower temp

in situ hybridization: full chromosomes or cells FISH

Question 9

microarray

Answer 9

thousands of sequences corresponding to each gene

clinical: genotyping
- which allele of a gene is present which determines what variant of a protein is being produced
- amplichip

gene expression profiling: mRNA to cDNA to RTPCR
-how much RNA is being made- mix cDNAs and incubate with chip. scan and record intensities at each spot