Taq Polymerase 47
Normally, it is a prerequisite for the PCR to know the sequences
flanking the region to be amplified. Different methods were developed
to circumvent this drawback: Circularization of DNA fragments and
subsequent cleavage in one known site result in a constellation, where
an unknown region is flanked by sequenced regions (64).
Normally, the PCR is applied to generate well-defined sequences.
Whole genome PCR involves the ligation of oligodeoxynucleotides
prior to the amplification, and constitutes a pool of material for further
manipulation (65). With this technique, possible pitfalls in microcloning
of dissected chromosome fragments could also be bypassed. After the
ligation of oligodeoxynucleotides to restriction fragments, the DNA
was amplified and cloned in high yields (66, 67).
Ligation of known sequences to chemically cleaved DNA templates
and subsequent PCR amplification allows a specific in vivo footprint
analysis (68) as well as detection of methylation patterns (69). The
principles of this ligation-mediated PCR application are depicted in
Fig. 6. Random cleavage sites are generated in genomic DNA using
Maxam-Gilbert chemistry. The strands are denatured, and a gene-
specific primer is annealed and extended with DNA polymerase.
Oligodeoxynucleotide linkers are blunt-end ligated to the double-
stranded products. The linker-ligated fragments are amplified with the
PCR and analyzed on sequencing gels.
4.4. Amplification of mRNA
The amplification of RNA is performed on a cDNA template pro-
duced by reverse transcriptase. A small and not very efficient reverse
transcriptase activity was detected in Taq polymerase (70). Molony
Murine Leukemia Virus (MMLV) reverse transcriptase requires buffer
conditions similar to Taq polymerase. Therefore, after addition ofpoly
dT or sequence-specific primers and reverse transcriptase, the PCR
can be executed directly in the same vial (71). Avian Myeloblastosis
Virus (AMV) reverse transcriptase and Taq polymerase may also be
added together to the RNA; the reactions will be started subsequently
by choosing the proper reaction temperatures (42).
Starting with cDNA, fragments as long as 1.6 kb could be cloned (72).
The extreme sensitivity of the PCR allows semiquantitative recordings on
specific mRNA content of single cells (73). Eleven out of 12 patients
suffering from chronic myelogenous leukemia (CML) who underwent
bone marrow transplantation still produced transcripts specific for CML.