Biology Reference
In-Depth Information
DNA decays spontaneously, mainly through hydrolysis and oxidation. Hydrolysis
causes deamination of the nucleotide bases and cleavage of base-sugar bonds,
creating baseless sites. Deamination of cytosine to uracil and depurination (loss of
purines adenine and guanine) are two types of hydrolytic damage. Baseless sites
weaken the DNA, causing breaks that fragment the DNA into smaller and smaller
pieces. Oxidation leads to chemical modification of bases and destruction of the
ring structure of base and sugar residues (
Austin 1997a
). As a result, it is almost
impossible to obtain long amplification products from ancient DNA (
Handt et al.
1994
). It is possible to use overlapping primer pairs if longer sequences are needed,
but there usually is an inverse relationship between efficiency and length of the PCR
products. When such an inverse relationship is
not
seen, the amplification product
often turns out to be due to contamination (
Handt et al. 1994, Hofreiter et al. 2001
).
PCR products from ancient DNA often are “scrambled.” This is due to the
phenomenon called “jumping PCR,” which occurs when the DNA polymerase
reaches a template position which carries either a lesion or a strand break that
stops the polymerase (
Handt et al. 1994
). The partially extended primer can
anneal to another template fragment in the next cycle and be extended up to
another damaged site. Thus,
in vitro
recombination (jumping) can take place
until the whole stretch encompassed by the two primers is synthesized and the
amplification enters the exponential part of the PCR (
Handt et al. 1994
). This
phenomenon makes it essential that cloning and sequencing of multiple clones
be carried out to eliminate this form of error.
Most archeological and paleontological specimens contain DNA from exog-
enous sources such as bacteria and fungi, as well as contaminating DNA from
contemporary humans (
Poinar and Stankiewicz 1999
). Aspects of burial condi-
tions seem to be important in DNA preservation, especially low temperature
during burial (
Poinar and Stankiewicz 1999
). The oldest DNA sequences reported
and confirmed by other laboratories are from the remains of a wooly mammoth
found in the Siberian permafrost; these sequences are “only” 50,000 years old—
not millions of years old (
Poinar and Stankiewicz 1999
).
More recently, ancient DNA has been sequenced using new Next-Generation
sequencing methods because small fragments of (degraded) DNA are sequenced
efficiently with these methods. Subsequent bioinformatic analysis allowed the sci-
entists to recognize and sort out the short bits of ancient DNA from longer pieces
of contaminant DNA. For example, the entire mammoth genome was sequenced
from DNA extracted from mammoth hair (
Miller et al. 2008
) and the entire
Neanderthal genome was sequenced in 2010 from DNA from bone fragments esti-
mated to be
≈
44,000 years old (
Green et al. 2010
). Thus, the PCR may not always
be the most appropriate method for analysis of ancient DNA.
