13 Sequencing
191
- Modified T7 DNA Polymerase (Sequenase™) (Tabor and Richardson
1987), shows a higher processivity, i.e.less premature termination of
chainelongationduetofall-offoftheenzyme,enablingreadingoflonger
sequences with less background. Furtheradvantages are reduced 3'- to5' exonuclease activity and more efficient incorporation of nucleotide
analogs.
- Taq Polymerase (Innis et al. 1988) (see also Chap. 14) is used at a temperatureof600 C. This allows ahigherstringencyofprimer hybridization
andadecreaseinsecondarystructureformationofthetemplatethatmay
otherwise Iead to artifacts on the gel. The high rate of polymerisation
adds to these advantages. The major practical advantage lies in the
thermal cycling of the sequencing reaction. By heating the reaction
to 95°C without inactivation of the enzyme, the hybrid of template
and new chain is denatured. This allows the template tobe used again
duringthe next sequencing cyclewhichleads to alinear amplification of
the formed chains and decreases the amount oftemplate required. This
process also increases the signal in tensityand decreases the sensitivi tyto
reaction conditions.
- Avian Myeloblastosis Virus Reverse Transcriptase (Karanthanasis
1982; Graham et al. 1986; Mierendorf and Pfeffer 1987) reads better
through GC-rich stretches than does Klenow fragment.
• Radiolabeled deoxynucleotide triphosphates (dNTPs)
a[ 32 P]dNTP or a[ 35 S]dNTP may be used. 35 S has the advantage ofhigh
resolution and operator safety (Biggin et al. 1983). At -21 o C it remains
stable for 4 to 6 weeks. There is, however, the disadvantage that gels have
to be dried prior to autoradiography.
• Reaction buffer
Mostenzymes workonlyunder appropriate electrolyte and pH conditions.
• Dideoxynucleotide and deoxynucleotide triphosphates ( ddNTPs and
dNTPs)
These reagents are available commercially in high concentrations. This
permitsthenecessaryadjustmentoftheconcentrationforeachsequencing
reaction. Secondary structures formed by DNA stretches with dyad symmetry, whichlead to compression bands on a sequencinggel, can usually be
resolved bythe use of nucleotide analogs such as diTP (inosine in place of
guanosine)and7-deaza-dGTP(Barnesetal.1983;GoughandMurray1983;
Mizusawa et all986; Innis et al. 1988). Theseare ideal substrates for Taq
polymerase and modified T7 polymerase.
191
- Modified T7 DNA Polymerase (Sequenase™) (Tabor and Richardson
1987), shows a higher processivity, i.e.less premature termination of
chainelongationduetofall-offoftheenzyme,enablingreadingoflonger
sequences with less background. Furtheradvantages are reduced 3'- to5' exonuclease activity and more efficient incorporation of nucleotide
analogs.
- Taq Polymerase (Innis et al. 1988) (see also Chap. 14) is used at a temperatureof600 C. This allows ahigherstringencyofprimer hybridization
andadecreaseinsecondarystructureformationofthetemplatethatmay
otherwise Iead to artifacts on the gel. The high rate of polymerisation
adds to these advantages. The major practical advantage lies in the
thermal cycling of the sequencing reaction. By heating the reaction
to 95°C without inactivation of the enzyme, the hybrid of template
and new chain is denatured. This allows the template tobe used again
duringthe next sequencing cyclewhichleads to alinear amplification of
the formed chains and decreases the amount oftemplate required. This
process also increases the signal in tensityand decreases the sensitivi tyto
reaction conditions.
- Avian Myeloblastosis Virus Reverse Transcriptase (Karanthanasis
1982; Graham et al. 1986; Mierendorf and Pfeffer 1987) reads better
through GC-rich stretches than does Klenow fragment.
• Radiolabeled deoxynucleotide triphosphates (dNTPs)
a[ 32 P]dNTP or a[ 35 S]dNTP may be used. 35 S has the advantage ofhigh
resolution and operator safety (Biggin et al. 1983). At -21 o C it remains
stable for 4 to 6 weeks. There is, however, the disadvantage that gels have
to be dried prior to autoradiography.
• Reaction buffer
Mostenzymes workonlyunder appropriate electrolyte and pH conditions.
• Dideoxynucleotide and deoxynucleotide triphosphates ( ddNTPs and
dNTPs)
These reagents are available commercially in high concentrations. This
permitsthenecessaryadjustmentoftheconcentrationforeachsequencing
reaction. Secondary structures formed by DNA stretches with dyad symmetry, whichlead to compression bands on a sequencinggel, can usually be
resolved bythe use of nucleotide analogs such as diTP (inosine in place of
guanosine)and7-deaza-dGTP(Barnesetal.1983;GoughandMurray1983;
Mizusawa et all986; Innis et al. 1988). Theseare ideal substrates for Taq
polymerase and modified T7 polymerase.