Biology Reference
In-Depth Information
25 ng template
Split peaks
1 ng template
Pull-up
1,000 RFU
(a)
(c)
0.25 ng template
2.5 ng template
Allele drop-out
Allele drop-out
2,000 RFU
50 RFU
(b)
(d)
Figure 7.5 If the reaction is overloaded with DNA (a) the peaks are still present but artefacts such
as pull-ups and split peaks are more pronounced. When the template is within the optimal range
(b and c) the peaks are well balanced and easy to interpret. When the PCR does not have enough
template to amplify (d) then locus and allelic drop-out can occur
Template DNA
Commercial STR kits have been optimized to amplify small amounts of template
DNA, commonly between 0.5 ng and 2.5 ng, which represents approximately 166
and 833 copies of the haploid human genome. It is not always possible to add the
optimum amount of DNA to a PCR when the sample size is limited.
Overloaded profiles
Overloading the PCR can also lead to a profile that is difficult to interpret. If the CCD
camera is saturated, then the peak height/area is no longer a good indicator of the
amount of product and this can lead to problems in assessing peak balance and can
make the interpretation of mixtures difficult. Overloaded profiles also tend to have a
noisy baseline, increased levels of stuttering, split peaks and pull-ups (Figure 7.6).
Low template DNA typing
At many crime scenes it may be possible to infer surfaces with which the perpetrator
has had physical contact, for example the handle of a gun, a knife, a ligature, a door
handle or a steering wheel. These areas can be swabbed to collect any epithelial
cells that have been shed during the contact [6 - 9]. The amounts of DNA extracted
can be extremely low but in some circumstances it is possible to get a full DNA
profile from less than 100 pg of template DNA: the normal range of template DNA is
between 500 pg and 2500 pg (2.5 ng). To analyse such small quantities of DNA the
 
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