Methods in Molecular Biology • 16 Enzymes of Molecular Biology

30 Maunders Molecular and Cell Biology Catalogue (1992/1993) FPLC pure T7 DNA Poly- merase, cloned. Pharmacia P-L Biochemicals ...

CHAPTER 4 Taq Polymerase (EC 2.7.7.7) With Particular Emphasis on Its Use in PCR Protocols Axel Landgraf and Heiner Wolfes Intr ...

32 Landgraf and Wollees The sequence for the enzyme allowed a better characterization of the Taq polymerase (5). Given the bioch ...

Taq Polymerase 33 Table 1 Inhibitor Effects on Taq Polymerase I Activity ~ Inhibitor Concentration % Activity Urea <0.5M 100 ...

34 Landgraf and Wolfes Polymerase Chain Reaction (PCR) The first publication concerning the polymerase chain reaction (PCR) app ...

Taq Polymerase 35 Cydel Cyde2 Cycle 3 t Dan~ure and anneal pdmere m m t Pdmer e0dention t Danalure and anneal pdmers emm m m m t ...

36 Landgraf and Wolfes log Primer incorporation Efllcienc, 2 1.8 1.6 1.4 1.2 1 0,8 Plateau phase f I I 1 I I I I 10 20 30 40 Cyc ...

Taq Polymerase 37 % primer incorporation 1,2 0,8 0,6 0,4 0,2 0 I1" I I I I 1 2 3 4 U taq polymerase Fig. 3. Dependency of primer ...

38 Landgraf and Wolfes from this limit, the yield of the reaction is restricted by too low enzyme concentration or by competitiv ...

Taq Polymerase 39 3.2.2. Primer In theory, at least a 16 bp of primer are necessary for an unambigu- ous hybridization to a give ...

40 Landgraf and Wolfes /N Standard Mismatch (32) Ruorescent label (26) Biotin label (14) G G A A T T C C GC - stretch (33) conse ...

Taq Polymerase 41 base mismatches. Therefore, new restriction sites can easily be intro- duced into an amplification product (32 ...

42 Landgraf and Wolfes 9O temp [*c] 70 50 Primer Template Denaturation Annealing / time [min] Fig. 5. Typical PCR temperature pr ...

Taq Polymerase 43 Temperature assessment in the heating block: (a) the time reading starts, when the desired value in the block ...

44 Landgraf and Wolfes Top this mixture carefully with paraffin oil, incubate at 95°C for 3 min, add 1-4 U of Taq polymerase/100 ...

Taq Polymerase 45 tissues of cancer metastases. We would like to restrict this chapter to some examples with characteristic feat ...

46 Landgraf and Wolfes Allele-specific oligodeoxynucleotide (ASO) PCR (57) is based on the fact that only fully complementary pr ...

Taq Polymerase 47 Normally, it is a prerequisite for the PCR to know the sequences flanking the region to be amplified. Differen ...

48 Landgraf and Wolfes Single strand breaks are generated In a footprintlng experiment or using Maxam Gilbert chemistry I Anneal ...

Taq Polymerase 49 of unknown sequences. This method is termed "anchor PCR" (75-77). An alternative to eDNA synthesis is a combin ...