Biology Reference
In-Depth Information
Table 6.1: Enzymes Useful for DNA Manipulation.
Enzyme type (name, source)
Functions in genetic engineering
Enzymes that synthesize DNA
DNA polymerase I (
E. coli
)
5
′
to 3
′
DNA synthesis of template DNA with a primer; exonuclease functions
(5
′
to 3
′
and 3
′
to 5
′
) used to correct errors in DNA synthesis
in vivo
;
generate labeled DNA probes by nick translation; synthesize cDNA
Klenow fragment of DNA
polymerase I (
E. coli
)
DNA synthesis without 5
′
to 3
′
exonuclease ability; makes ds DNA from ss
DNA; used in dideoxy sequencing; DNA labeling by random priming or
end filling; converts 5
′
overhangs of DNA cut with restriction enzymes to
blunt ends; removes 3
′
overhangs to form blunt ends
T4 DNA polymerase (phage T4)
Exonuclease in 3
′
to 5
′
direction; fill in overhanging ends of DNA cut with
restriction enzymes
T7 DNA polymerase (phage T7)
3
′
to 5
′
exonuclease activity used in DNA labeling; converts 3
′
overhangs to
blunt ends
Taq
DNA polymerase (
Thermus
aquaticus
)
DNA synthesis at 60-70 °C in PCR; several cloned versions are available, as
are DNA polymerases from other microorganisms
Pfu
DNA polymerase (
Pyrococcus
furiosis
)
This polymerase and others have 3
′
to 5
′
exonuclease activity that allows
them to remove mismatches; used to amplify DNA fragments up to 40 kb
by the PCR
Reverse transcriptases (from
several RNA tumor viruses)
Synthesize single strand of DNA from messenger RNA; used to produce cDNA
libraries
Terminal transferase
(mammalian thymus)
Adds residues to any free 3
′
terminus; used to add poly(dG) and poly(dC) to
two DNA molecules to be joined
Enzymes that degrade DNA
S1 nuclease (
Aspergillus
)
Degrades ss DNA endonucleolytically; removes projecting 3
′
regions of ss
DNA in cloning and S1 mapping
Exonuclease III (
E. coli
)
Degrades one of two strands of ds DNA from 3
′
end of a blunt-ended double
helix or from a projecting 5
′
end
Bal 31 nuclease (
Alteromonas
espejiana
)
Degrades both strands of ds DNA with blunt ends
DNase I (pancreas)
Introduces random nicks in ds DNA before labeling by nick translation;
produces random fragments for shotgun cloning and sequencing in M13;
study chromatin structure; study DNA-protein complexes
Enzymes that join DNA
T4 DNA ligase Seals ss nicks between adjacent nucleotides in ds DNA molecule, requires
ATP; used to ligate two restriction fragments of DNA together in cloning
E. coli
DNA ligase Same as for T4 DNA ligase, but requires NAD
+
Enzymes that modify the 5
′
end
s of DNA
Calf intestinal phosphatase
CIP removes 5
′
-phosphate groups to generate a OH terminus; prevents
unwanted ligation of DNA fragments during cloning; used for end-labeling
DNA probes
T4 polynucleotide kinase
Adds phosphates to 5
′
-OH ends; used in chemical cleavage method of DNA
sequencing; used to add linkers or adapters in cloning
Enzymes that cut DNA
Restriction endonucleases
(many bacteria)
Type I, II, and III,
>
1400 types known; cleaves DNA, produces predictable
termini, either blunt, 5
′
overhang, or 3
′
overhang