This document discusses mixed DNA samples and low copy number (LCN) DNA analysis. It explains that mixed DNA samples contain DNA from multiple contributors, complicating analysis. LCN analysis involves increasing PCR cycles to enhance detection of very small amounts of DNA, but this can cause stochastic effects like allele dropout. The document discusses various techniques for analyzing mixed DNA samples, including using highly polymorphic markers, obtaining quantitative peak data, distinguishing genotypes, and statistical approaches. It also explains issues that can arise with LCN analysis, such as allele dropout and drop-in from contamination.

1. BY PANKAJ JANGRA
MIXED SAMPLE
AND LOW COPY
NUMBER OF DNA
MSc forensic science 9th semester

2. Mixed DNA contains DNA from two or more
contributors, compounding DNA analysis by combining
DNA from one or more major contributors with small
amounts of DNA from potentially numerous minor
contributors.
MIXED SAMPLE
Low copy number (LCN) analysis, also known as low
template DNA (LT-DNA) testing, involves enhancing
detection sensitivity usually through increasing the
number of PCR cycles. Stochastic effects inherent
with analysis of low amounts of DNA yield allele or
locus drop-out.
LOW COPY NUMBER

3. 1. VALUE OF HIGHLY POLYMORPHIC MARKERS
IN NON CLEAR MIXTURES
MIXED SAMPLE
Various techniques are used that increase the sensitivity
of a mixed samples.
In this method using highly polymorphic STR markers
with more possible alleles translates to a greater chance
of seeing differences between the two components of a
mixture. For example, D18S51 has 51 possible alleles while
TPOX only has 15 known alleles, making D18S51 a more
useful marker for detecting mixtures.

4. 2. QUANTITATIVE INFORMATION FROM
FLUORESCENCE MEASUREMENTS
The ability to obtain quantitative
information from peaks in an
electropherogram,using the ABI
310 or 3100 platform, permits
relative peak heights or areas of
STR alleles to be measured. This
peak information can then be used
to decipher the possible genotypes
of the contributors to the mixed
sample.

5. 3. DISTINGUISHING GENOTYPES IN A MIXED
SAMPLE
In which following questions can help ascertain the
genotypes that make up the composite DNA profile of the
mixture:
■ Does any of the loci show more than two peaks in the
expected allele size range?
■ Is there a severe peak height imbalance between heterozygous
alleles at a locus?
■ Does the stutter product appear abnormally high (e.g., 15% to
20%)?

6. If the answer to any one of these three questions is ‘
yes, ’then the DNA profile may very well have resulted
from a mixed sample.
Usually a mixture is first identified by the presence of
three or more prominent peaks at one or more loci . At a
single locus, a sample containing DNA from two sources
can exhibit one, two, three, or four peaks due to the
possible genotype combinations .

7. The six primary steps for interpreting mixtures
1. Identify the Presence of mixtures.
2. Designate Allele Peaks.
3. Identify the numbers of potential contributor.
4. Identify the relative ratio of individual contributing to
the mixtures.
5. Consider All Possible Genotype Combinations.
6. Compare refrences sample.
MIXTURE INTERPRETATION

8. Three different approaches are generally taken with
statistical analysis of mixtures:(1) performing match
probability estimates on the major contributor after
deducing the possible genotypes of the contributors using,
for example, the strategy outlined in the previous section;
(2) calculating exclusion probabilities and (3) performing
likelihood ratio calculations.
STATISTICAL APPROACHES TO MIXTURE
ANALYSIS

9. LOW COPY NUMBER
Many laboratories have demonstrated the ability to
obtain DNA profiles from very small amounts of sample.
Low copy number (LCN) DNA testing is referred to as low-
level DNA, basically its refers to examination of dna sample
is less than 100 pg .
INCREASE THE NUMBERS OF PCR CYCLES
The number of PCR cycles is often increased to improve
the amplification from samples containing extremely
low levels of DNA template

10. In which touch DNA are the major sample source for
the LCN DNA profiles may be obtained from
fingerprint residues due to the cells that are left on
the objects while touch the object.
In which increasing the PCR cycle number from 28 to
34, for more copies of DNA molecules are
generated. With 100% efficiency, while 28 cycles of
PCR generate 4 billion copies of DNA.

11. When LCN testing is performed, three major issues are
observed.
(1) additional alleles are often observed from sporadic
contamination in what is referred to as allele ‘ drop-in, ’.
(2) allele ‘dropout ’is common where an allele fails to
amplify due to stochastic effects.
(3) stutter product amounts are enhanced so that they are
often higher than the typical 5% to 10% of the nominal
allele. Heterozygote peak imbalance is typically
exacerbated due to stochastic PCR amplification.
ISSUES WITH LCN WORK