Methods in Molecular Biology • 16 Enzymes of Molecular Biology

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)

consensus sequence (34,35)

Fig. 4. Versatility of PCR primers.

Apparently, primer pairs are formed and elongated by the enzyme (24).
Biased primer concentration results in asymmetric amplification of one
strand (25) (Section 4.3.).
PCR is capable of dealing with many different problems by utilization
of primers specifically designed for a particular purpose (Fig. 4). Primers
carrying a chemical label at the 5' end, e.g., oligodeoxynucleotides sub-
stituted with a biotin or fluorescent dye, are well accepted by Taq poly-
merase. Oligodeoxynucleotides can be synthesized with a primary amino
group (aminolink) at the 5' end, to which biotin (14) or fluorescent mark-
ers (26) may be coupled. As a consequence, 5' biotinylated primers offer
the option to exploit avidin-coupled dye reactions as well as the immobi-
lization of PCR products via an avidin (14) or streptavidin bridge (27) to
a matrix. The use of fluorescent primers offers a variety of applications in
qualitative and quantitative analysis, e.g., nonisotopic sequencing reac-
tions (28,29) or color coupling tests (30).
A perfect homology to the target sequence is not a conditio sine qua
non for primers, although a high degree of sequence homology is
necessary. Taking into account that the enzyme is working down-
stream from the 3' end of the primer, only the last three bases adjacent
to this region need to be correctly base paired, in order to initiate
polymerization (31). The 5' region of the primer is less sensitive to