A description of Genetic Markers and their applications with focus on Forensic Analysis. Complimentary methods such as RNA Profiling are also discussed.

1. Importance of Genetic Markers
in Forensic Analysis
Mrinal Vashisth
B.Tech. – Biotechnology
6-BT-12

2. What are Genetic Markers?
• A gene or DNA seq. with a known location on the chromosome
• Single base pair changes such as SNPs and minisatellites act as
common genetic markers
• These can be of two types:
a) biochemical markers which detect variation at the translational
level e.g. amino acid and protein changes
b) molecular markers which detect variation at the DNA level such
as nucleotide changes: duplication, inversion, INDELs etc.
Uses of Genetic Markers
• Used for individual or species identification purposes
• Study relationship between disease and its cause

3. Types of Genetic Markers
• RFLP (or Restriction fragment length polymorphism)
with PCR
• SSLP (or Simple sequence length polymorphism) with
PCR
• RAPD (or Random amplification of polymorphic DNA)
• VNTR (or Variable number tandem repeat)
– SSR Microsatellite polymorphism, (or Simple sequence
repeat)
– SNP (or Single nucleotide polymorphism)
– STR (or Short tandem repeat)
• AFLP (or Amplified fragment length polymorphism)
with PCR

4. Basic Principle
• 40% of human genome is repetitive DNA
• These were first identified in Satellite DNA
• When a genome is digested with REs (e.g. EcoRI)
– it is seen that there are repeats throughout the
genome
• Some such repeats are actually clustered at
specific locations -- tandem repeats (more
common) and inverted repeats
• VNTRs are one of the class of clustered tandem
repeats that exhibit allelic variation in their
lengths.

5. Restriction Fragment Length
Polymorphism (RFLP)
• Difference in
homologous DNA
sequences that can be
detected by the
presence of fragments of
different lengths after
digestion of the DNA
samples in q
• Co-dominant (both
alleles in heterozygous
sample will be detected)
and highly locus-specific
with specific restriction
endonucleases.

6. Simple Sequence Length
Polymorphisms (SSLPs)
• SSLPs are repeated sequences over varying base lengths
in intergenic regions of deoxyribonucleic acid (DNA)
• Can be used to compare individual differences
Membership probability of
assigning genotypes of the
entire population to
(a) two, (b) three, (c) four,
(d) five subgroups. The
height of each bar
represents
(b) the probability of
varieties belonging to
different subgroups.
The varieties
(c) were sorted according
to their membership
probability in (a).

7. Random Amplification of Polymorphic
DNA (RAPD)
• Add primers (8-12) nt long to
the DNA sample and PCR
amplify it
• Resolve the resulting pattern
• Primers would bind to
unknown sequences at
unknown sites thus there is no
need to have an existing
comparative database
• Degraded DNA samples are not
used
• Used to trace phylogenies
• There are problems in
reproducibility of results, many
scientific journals do not
accept experiments merely
based on RAPDs anymore
A silver-stained polyacrylamide gel showing
three distinct RAPD profiles generated by
primer OPE15 for Haemophilus ducreyi isolates
from Tanzania, Senegal, Thailand, Europe, and
North America
Different species produce different bands
Nearly all RAPD markers are dominant

8. Variable number tandem repeat
(VNTR)
• The tandem repeats in various
genes can be used for personal
and parental identification
• The key is: repeats are
inherited and people differ in
terms of repeats – children
and parents match in terms of
inheritance of repeating
pattern (which is unique for
each individual)
• The superset of parents’ VNTR
contains children’s pattern as a
subset and the grandparents’
pattern would be related to
grandchildren in the same
sense
Variations of VNTR (D1S80) allele
lengths in 6 individuals

9. • There are two types of VNTRs:
• 1.) Microsatellites: Near about 5 bps
– 2 nt differences among tissues in an individual
– 3 nt differences – across generations
– CODIS uses 13 assay STR with 4 nt differences for
forensic purpose
• 2.) Minisatellites: > 5 bps
– Short Tandem Repeat (STR) are sort of
minisatellites used in Forensic Analysis by Forensic
Geneticists
– Simple Sequence Repeat (SSR) are basically the
same but the term is more prevalent among Plant
Geneticists – these give more crucial information

10. Amplified fragment length
polymorphism (AFLP)
• Although AFLP is commonly
referred to as "Amplified
fragment length polymorphism",
the resulting data are not scored
as length polymorphisms, but
instead as presence-absence
polymorphisms.
• Used in criminal and paternity
tests
• To determine slight differences
within population
• Linkage studies to generate maps
for quantitative trait locus (QTL)
analysis
• Highest reproducibility,
sensitivity and resolution at the
whole genome level out of the
discussed techniques

11. Forensic Analysis
• The first use of DNA fingerprinting was seen in 1986’s Dawn Ashworth
case in the UK
• Most human forensic casework is performed with standardized
commercial “multiplexes” that assay STRs at multiple genetic loci
simultaneously
• In the United States, the DNA Identification Act of 1994 authorized the FBI
to create a national DNA database: the Combined DNA Index System
(CODIS) -- contains profiles of DNA samples taken from convicted
offenders
• Criminal investigators can query CODIS with STR profiles taken from
biological samples found at a crime scene -- consists of 13 STR loci plus
the amelogenin gene
• Laws regarding whose DNA goes into the database varies from country to
country
• In some countries, investigators now search DNA databases not only for
full matches but also for partial matches to STR profiles
STR profile

12. • Researchers are now establishing useful markers
on the Y chromosome – to screen out victim of
sexual assault versus the convict
• Other specialized markers such as miniSTRs for
which SNP profiles were developed to identify
the victims of September 11, NYC terrorist attack
• The main disadvantage of SNP: not many
varieties as compared to STRs
• Almost 50 SNPs are required as compared to 13
STRs
• mtDNA is useful in tracing the maternal lines and
thus determining the origin (the famous
Romanov family case) – historical analysis of
bones and in cells with little DNA

13. Complimentary Methods
• DNA profiling says nothing about
the tissue type which can be
determined by RNA profiling
• Messenger RNA markers have also
been identified for saliva, semen,
and vaginal secretions – this sort
of Biological fluid analysis screens
out the victim’s DNA
• Recent work in human genetic
variation suggests that researchers
may be able to extract information
about a person's ancestry from
certain key genetic markers
• A major challenge in this area is
finding markers that can deal with
heavily admixed populations, or
those in which people of very
different ancestries have blended
together
Consensus neighbour-joining tree
of the 249 non-admixed human
populations and six chimpanzee
populations

14. Conclusion
• Genetic markers such as VNTRs can be used for
identification purposes
• These marker techniques combined with PCR can be
used to study minute amount of DNA samples present
at crime site
• STR analysis of 13 sites and 1 gene forms a DNA
fingerprint which can be compared against CODIS or
similar databases
• In case of highly damaged DNA we miniSTR and SNP
analysis provides a useful basis of identification (at
ethnic level in un-admixed populations)
• Y chromosome, mtDNA, RNA profile all add on to the
information one can obtain from a given forensic
sample

15. Thank you!

16. References
• Zhang P, Li J, Li X, Liu X, Zhao X, Lu Y (2011) Population Structure and
Genetic Diversity in a Rice Core Collection (Oryza sativa L.)
Investigated with SSR Markers. PLoS ONE 6(12): e27565.
doi:10.1371/journal.pone.0027565
• rDNA: Random Amplification of Polymorphic DNA (RAPD).
www.rvc.ac.uk. Retrieved 09-04-2017
• Trevor J P, Michael DG and Noah A. Rosenberg - Trevor J. Pemberton
et al. Population Structure in a Comprehensive Genomic Data Set
on Human Microsatellite Variation. G3 (Bethesda). 2013 May
20;3(5):891-907. doi: 10.1534/g3.113.005728
• Phillips M L, Crime Scene Genetics: Transforming Forensic Science
through Molecular Technologies, BioScience (2008) 58 (6): 484-489
, https://doi.org/10.1641/B580604