Biology Reference
In-Depth Information
to decipher the sequence of individual alleles (
Rao 1994a
). Under these circum-
stances, the amplified DNA should be cloned and the sequences of many clones
should be determined to resolve the sequences of individual alleles.
Direct sequencing of PCR-amplified DNA is inappropriate if nonspecific prod-
ucts were produced. Thus, direct sequencing requires good PCR conditions: qual-
ity DNA template, highly specific primers, high annealing temperature, initiating
the PCR by the “hot-start” method, and performing a rapid cycling protocol for
as few cycles as possible using an adequate amount of target DNA (
Rao 1994a
).
8.6 Multiple Displacement Amplification: Another Method to
Amplify DNA
Multiple displacement amplification (MDA) is a non-PCR-based DNA amplifica-
tion method that can amplify very small amounts of DNA, even from a single
cell (
Cheung et al. 1996, Dean et al. 2001, 2002, Gorrochotegui-Escalate and
Black 2003, Hosono et al. 2003, Jeyapakash and Hoy 2004, Panelli et al. 2006
).
After amplification by MDA, the PCR can be used to further amplify specific
genes. MDA can amplify genomic DNA within a few hours without a thermal
cycler at 30 °C using exonuclease-resistant thiophosphate-modified hexam-
ers (random nucleotides) as primers and bacteriophage Phi29 DNA polymerase,
which is a high-fidelity enzyme. It is thought that the hexamers bind at random
over the genome, allowing Phi29 DNA polymerase to synthesize DNA strands up
to 7-10kb, enriching each amplified DNA strand by approximately 10,000-fold
in ~3 hours (
Dean et al. 2001, 2002, Hosono et al. 2003, Tranah et al. 2003
).
MDA can be used to amplify DNA from single adult legs or larvae of a mos-
quito, producing sufficient DNA for subsequent amplification by the PCR or
analysis by the Southern blot (
Gorrochotegui-Escalante and Black 2003
). Whole
genome amplification (WGA) can be used to amplify microbial DNA from arthro-
pods;
Jeyaprakash and Hoy (2004)
produced sufficient DNA from
Wolbachia
or
Cardinium
bacteria in a predatory mite egg that subsequent amplification by
high-fidelity PCR was successful. A sensitivity analysis suggested that WGA fol-
lowed by the high-fidelity PCR was able to detect as little as 0.01 fg of the target
DNA, equivalent to about one copy.
Kumar et al. (2008)
found that MDA amplifi-
cation of single human cells resulted in high-quality DNA with few artifacts.
8.7 Concluding Remarks
The speed, specificity, versatility, and sensitivity of the PCR have had a significant
effect on genetics, immunology, forensic science, evolutionary biology, systemat-
ics, ecology, and population biology (
Arnheim and Erlich 1992, Dieffenbach 1995
).
