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KAI-OLAF NETZER
I Subprotocol 6
T 4 and T7 DNA Polymerase
Bacteriophage T4 and T7 encoded DNA polymerase are similar to the Klenow fragment of E. coli polymerase in that they possess 5' ~3' polymerase
activity and 3' ~s' exonuclease (proofreading) activity. However, the T4
and T7 enzymes are 200 fold and 1000 fold, respectively, more active (faster)
than the Klenow fragment. One of the uses ofT 4 polymerase is the conversion of double stranded DNA with protruding 3' -OH ends to blunt ended
molecules (see below). T4 polymerase can also convert protruding 5' -OH
ends to blunt ended molecules by using 5'~3' polymerase activity.
The primary use ofT? DNA polymerase is in primer extension reactions
that require the copying of long stretches of template, such as sequencing
reactions. Forthis purpose, a modified T7 DNA polymerase (Sequenase™)
lacking the 3' ~s' exonuclease activity is commercially available.
Removing protruding 3' termini
The 3'~5' exonuclease activityofT4 DNA polymerase removes protruding
3' termini until it reaches the second strand. Further exonucleolytic activity
is then balanced by incorporation (5'~3' polymerase activity) of dNTPs
which have tobe added to the reaction. T4 DNA polymerase is preferred
over Klenow enzyme due to its 200-fold higher activity.
Materials
Supplies
• Cooling water bath l2°C, water bath 75°C
Reagents
and solutions
• Alternatively: Thermal cycler, programmed to l2°C/30 min, 75°C/10
min, maximum temperature slope
• DNA (O.l-SJlgin 20 Jll) whichhas been digested withrestriction enzymes
that create protruding 3' ends. T4 polymerase has about 50% of its maximum activity in all commonly used restriction enzyme buffers. Therefore, the reaction can be performed after DNA digestion in the same reaction medium. Alternatively, restriction enzyme digests may be purified
with chaotropic buffer/silica-gel spin columns before the reaction.
KAI-OLAF NETZER
I Subprotocol 6
T 4 and T7 DNA Polymerase
Bacteriophage T4 and T7 encoded DNA polymerase are similar to the Klenow fragment of E. coli polymerase in that they possess 5' ~3' polymerase
activity and 3' ~s' exonuclease (proofreading) activity. However, the T4
and T7 enzymes are 200 fold and 1000 fold, respectively, more active (faster)
than the Klenow fragment. One of the uses ofT 4 polymerase is the conversion of double stranded DNA with protruding 3' -OH ends to blunt ended
molecules (see below). T4 polymerase can also convert protruding 5' -OH
ends to blunt ended molecules by using 5'~3' polymerase activity.
The primary use ofT? DNA polymerase is in primer extension reactions
that require the copying of long stretches of template, such as sequencing
reactions. Forthis purpose, a modified T7 DNA polymerase (Sequenase™)
lacking the 3' ~s' exonuclease activity is commercially available.
Removing protruding 3' termini
The 3'~5' exonuclease activityofT4 DNA polymerase removes protruding
3' termini until it reaches the second strand. Further exonucleolytic activity
is then balanced by incorporation (5'~3' polymerase activity) of dNTPs
which have tobe added to the reaction. T4 DNA polymerase is preferred
over Klenow enzyme due to its 200-fold higher activity.
Materials
Supplies
• Cooling water bath l2°C, water bath 75°C
Reagents
and solutions
• Alternatively: Thermal cycler, programmed to l2°C/30 min, 75°C/10
min, maximum temperature slope
• DNA (O.l-SJlgin 20 Jll) whichhas been digested withrestriction enzymes
that create protruding 3' ends. T4 polymerase has about 50% of its maximum activity in all commonly used restriction enzyme buffers. Therefore, the reaction can be performed after DNA digestion in the same reaction medium. Alternatively, restriction enzyme digests may be purified
with chaotropic buffer/silica-gel spin columns before the reaction.