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Flashcards in Techniques in Molecular Medicine Deck (16):

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

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


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

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


Restriction endonucleases

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

Break down foreign DNA


How does and restriction enzyme choose from host and foreign?

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


Properties of restriction enzyme

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)


Genomic DNA

Identical in all cells of an organism
Typically isolated from lymphocytes


Gel electrophoresis- Blots

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



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


Probes can be made from:

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



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


Stringency of Hybridization

Can be controlled by modifying temperature at which reaction occurs

higher stringency= higher temperature



Restriction Fragment Length Polymorphism
Restriction enzyme cuts DNA at specific sequences



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


Taq Polymerase

Thermal resistent DNA polymerase for PCR


Steps of PCR

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*


PCR in Forensics

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