BIOL 311 Human Genetics
Fall 2006
Lecture: Recombinant DNA Methods—DNA and RNA probes
Reading: Chapter 6 pp. 136-161
Lecture Outline:
1. Probes
2. Labeling DNA:
a. Nick translation
b. Random primed DNA labeling
c. Riboprobes
d. End-labeling
e. non-radioactive labeling detection
Lecture:
1. Probes: labeled fragments of DNA, oligonucleotides or RNA strands that are mainly
used in hybridization experiments (Southerns, Northerns, microarrays, etc.) to detect a
sequence of interest.
2. Labeling DNA: attach radioactive or non-radioactive tag to make it easier to track
small quantities of DNA (on the order of femtomoles—10-15 moles).
Main approaches:
--uniformly labeled DNA—attach many tag molecules
--end-labeling—attach one or two tag molecules to end of DNA strand
Mostly “in vitro” approaches involving enzymes that add the tags to the DNA in a small
biochemical reaction.
a. Nick translation—Use E. coli DNA polymerase (+/- DNase I) to add radioactive
nucleotides at a nick. Can be used on circular or linear DNAs.
E.coli DNA polymerase: Main DNA repair enzyme of E. coli (Kornberg’s first enzyme)
3 functions: 5’ 3’ DNA polymerase
3’5’ exonuclease (proofreading function)
5’3’ exonuclease (repair function, impt. For nick translation.
Typical reaction:
DNA template
Buffer + Mg2+
DNase I (brief incubation , then inactivate)
DNA polymerase I (Holoenzyme, NOT Klenow)
dNTPs, including at least one radioactively labeled, usually with 32P
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b. Random primed DNA labeling—use short oligonucleotide primers of random
sequence to prime DNA synthesis on a single-stranded or denatured double-stranded
DNA template in the presence of a tagged nucleotide (can be radioactive).
Uses DNA polymerase I Klenow fragment—portion of DNA polymerase containing
5’3’ DNA polymerase activity and 3’ 5’ exonuclease activity (proofreading
function).
Typical reaction:
Denatured DNA template
Random primers
Buffer + Mg2+
DNA polymerase I Klenow (NOT holoenzyme)
dNTPs, including one radioactively labeled, usually with 32P
c. Riboprobes—labeled RNA probes, usually synthesized in vitro from a DNA template
containing a promoter recognized by an E. coli viral RNA polymerase, i. e. T7, T3, Sp6.
Uses a viral RNA polymerase to synthesize RNA 5’3’ from a DNA template.
Typical reaction: T7 riboprobe
Linearize DNA template attached to T7 phage promoter
T7 RNA polymerase
Buffer
RNasin (inhibitor of RNase)
NTPs, including one labeled (often 32P-CTP)
d. End-labeling—Attach radioactive nucleotide or radioactive phosphorus to end(s) of a
DNA molecule.
Examples:
Klenow end labeling of 5’ overhangs generated by restriction enzymes.
Kinase labeling of 5’ ends of oligonucleotides
e. non-radioactive labeling detection--Attach molecule to DNA that can be detected in
a chemical reaction that produces a light emitting or colored product.
Examples: Biotin/streptavidin-alkaline phosphatase or horse radish peroxidase
Digoxigenin/antibody to digoxigenin linked to enzyme
Fluorophores (fluorescent dyes) can be attached to streptavidin or to
antibodies to create fluorescent labels for microarrays.
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