Department of Biology
Memorial University of Newfoundland
Other Enzymes in Molecular Techniques
Polymerases
1) Thermophilic DNA polymerases and PCR
2) T7 DNA polymerase for second-strand synthesis in site-directed mutagenesis.
3) DNA polymerase I (E. coli) for nick translation and for second-strand
synthesis in site-directed mutagenesis.
4) DNA polymerase I: Large Fragment (Klenow Fragment) no 5 -> 3 prime
exonuclease activity used for
a) fill-in of 5 prime overhangs to generate blunt
ends
b) removal of 3 prime overhangs to generate blunt
ends
c) second-strand cDNA synthesis.
d) second-strand synthesis in site-directed mutagenesis.
5) Klenow Fragment without 3 -> 5 prime exonuclease activity used for
a) generating probes from random priming labeling
b) second-strand cDNA synthesis.
c) second-strand synthesis in site-directed mutagenesis
6) T4 DNA polymerase has no 5 -> 3 prime exonuclease activity used
for
a) fill-in of 5 prime overhangs to generate blunt
ends
b) removal of 3 prime overhangs to generate blunt
ends
c) second-strand synthesis in site-directed mutagenesis.
7) M-MuLV reverse transcriptase is used to polymerase DNA from an RNA
template (or single-stranded DNA)
8) Terminal transferase is used to add a homopolymer tail to the 3
prime end of DNA molecules and can be used to lable the end of DNA
9) T7 and SP6 RNA polymerases generate RNA from a DNA template and
can be used to
a) labeled RNA probes
b) generate RNA for in vitro translation
c) generate RNA for studies of RNA functions
d) generation of anti-sense and double stranded
RNA
DNA and RNA modifying enzymes
1) Methylases can block restriction endonuclease activity/increase
(practical) specificity of restriction endonuclease and can be used to
study methylated dependant activity.
2) DnaseI is used to
a) remove DNA template in in vitro transcription
reactions
b) remove contaminating DNA in RNA preparations
c) DnaseI footprinting
d) nick translation
3) Exonuclease I degrades single stranded DNA (primer/oligonucleotides)
in a mixure containing double-stranded DNA
4) Exonuclease III digests a single strand of a double-stranded DNA
but not single-stranded DNA and is used
a) to generate nested deletions and
b) in site-directed mutagenesis.
5) RnaseH can be used to remove mRNA in cDNA synthesis and poly A tails
(when hybridized to poly dT
6) Rnases in RNA protection assays and general removal of RNA.
7) T4 DNA ligase is used to join restriction fragments and vectors
and to join linkers to blunt-ended DNA
8) T4 RNA ligase is used to lable 3 prime ends of RNA, to synthesize
single-stranded rna oligonucleotides and to ligate single-stranded RNA
(and DNA hybrids)
9) T4 polynucleotide kinase can add 5 prime phosphates and remove 3
prime phopshates and is used to lable the 5 prime end of DNA and RNA.
10) Beta -agarase digests agarose to release DNA fragments.
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