Techniques in Molecular Medicine

Question 1

To identify differentially expressed genes in tumor and normal tissue, you could isolate ____

Answer 1

RNA or proteins (b/c we’re looking at differentially expressed genes)

Question 2

To identify mutations causing altered splice sites, you could isolate ______

Answer 2

Genomic DNA (b/c looking at structure of gene where the mutation occurs, RNA will not give info about structure of gene)

Question 3

Restriction endonucleases

Answer 3

Bacterial enzymes that cleave dsDNA and are a specific sequence (NOT RNA!!!)

Break down foreign DNA

Question 4

How does and restriction enzyme choose from host and foreign?

Answer 4

Host DNA not broken down b/c for each restriction enzyme there is a corresponding methylase that adds a methyl group to the restriction enzyme sites on the bacterial genome to protect them from cleavage

Question 5

Properties of restriction enzyme

Answer 5

1) Enzymes evolved to protect from invading organisms- named according to host
2) Recognition sites are 4, 6, 8 bases long
3) All sites palindromes over 2 strands
4) Enzymes can generate blunt/sticky ends (3’ or 5’ overhand)

Question 6

Genomic DNA

Answer 6

Identical in all cells of an organism

Typically isolated from lymphocytes

Question 7

Gel electrophoresis- Blots

Answer 7

DNA, RNA, and protein can be separated by gel electrophoresis to resolve different species of fragments by size

DNA (cut by restriction enzymes) and RNA are typically electrophoresed on agarose gels

Prteins on polyacrylmide gels

Question 8

Probes

Answer 8

Piece of ssDNA of a known sequence used to identify specific DNA and RNA on blots

Question 9

Probes can be made from:

Answer 9

1) Synthetic oligonucleotides
2) Restriction fragments of genomic DNA
3) cDNA made from RNA using reverse transcriptase
4) Fragments produced by PCR

Question 10

Hybridization

Answer 10

Probe is used to identify DNA or RNA of complementary sequence on the blot
^both must be single stranded

Question 11

Stringency of Hybridization

Answer 11

Can be controlled by modifying temperature at which reaction occurs

higher stringency= higher temperature

Question 12

RFLP

Answer 12

Restriction Fragment Length Polymorphism

Restriction enzyme cuts DNA at specific sequences

Question 13

PCR

Answer 13

Polymerase Chain Reaction

Ability to rapidly make multiple copies of defined pieces of (ds) DNA

Question 14

Taq Polymerase

Answer 14

Thermal resistent DNA polymerase for PCR

Question 15

Steps of PCR

Answer 15

1) Separation of template DNA strands by heating (95)
2) Annealing 2 DNA primers that flank the region to be amplified (50)
3) DNA synthesis catalyzed by thermostable DNA polymerase (72)
4) Repeat 30 times
* exponential increase*

Question 16

PCR in Forensics

Answer 16

Human genome DNA has repetitive sequences in tandem a variable # of times (VNTR)

Repeat units can be 2-60 bp long and can be copied 2-40 times

Short tandem repeats analyzed to establish identity
A given STR will occur at the same position in the genomic DNA of all individuals so each will have the same flanking sequence
Primers complementary to these flanking sequences can be used to amplify the repeat units by PCR