SUBJECT FORENSIC SCIENCE
Paper No. and Title PAPER No.13: DNA Forensics
Module No. and Title MODULE No.14: Short Tandem Repeats
Module Tag FSC_P13_M14
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats
TABLE OF CONTENTS
1. Learning Outcomes
2. Introduction
3. Forensic Issues
4. Types of Short Tandem Repeats Y-STR
5. Y-STR
6. Mini-STR
7. Characteristics of STRs selected for Human Identification
8. Advantages of STRs over conventional VNTRs
9. Applications of STRs
10. Summary
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats
1. Learning Outcomes
After studying this module, reader shall be able to understand -
Short tandem repeats (STR) and their forensic importance
Advantage of STRs over conventional VNTRs and applications of short tandem
repeats
2. Introduction
Genomes of eukaryotes are full of repeated DNA sequences. These sequences are
present in various sizes. They are usually named according to the length of the core
repeat unit and the number of adjacent repeat units or complete length of the repeat
region. There might be several hundred to thousand bases in core repeats of long
repeat units. Those regions of DNA which have short repeats (2-6 bps in length) are
known as Short Tandem Repeats (STRs). These are highly polymorphic
microsatellites. The center of the chromosome is surrounded by STRs.
Thousands of polymorphic microsatellites have been discovered in human DNA.
Only 3% of human genome comprises of Short Tandem Repeats. These are dispersed
throughout the genome and are present after every 1000 nucleotides on the average.
Figure 1: 13 CODIS core STR loci with chromosomal positions
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats
3. Forensic Issues
Figure 2: Comparison of crime scene and suspect DNA
4. Types of Short Tandem Repeats
Types of short tandem repeats on the basis of length:
i. Dinucleotides: They have two nucleotides which are repeated next to each
other again and again.
ii. Trinucleotides: They have three nucleotides which are repeated next to each
other again and again.
iii. Tetranucleotides: They have four nucleotides which are repeated next to each
other again and again.
iv. Pentanucleotides: They have five nucleotides which are repeated next to each
other again and again.
v. Hexanucleotides: They have six nucleotides which are repeated next to each
other again and again.
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats
Types of short tandem repeats on the basis of pattern:
i. Simple repeats: Identical length and sequence are present in simple repeats.
ii. Compound repeats: Two or more adjacent simple repeats are present in
compound repeats.
iii. Complex repeats: Several repeat blocks of variable unit length and variable
intervening sequences are present in complex sequences. Complex repeats are
not commonly used in forensic DNA typing because of the difficulties with
measurement variability between laboratories and allele nomenclature.
5. Y-STR
These are Short Tandem Repeats present on male specific Y chromosome. Short arm
of Y chromosome comprises of coding genes which are responsible for
spermatogenesis and other male related functions and in determination of male sex.
Among unrelated males, these Short Tandem Repeats are polymorphic. They are
inherited from father (paternal line) and show little change through generations. They
are mainly used to examine sexual assault evidence in forensic laboratories. In such
cases female as well as male DNA will be present in vaginal swabs. To separate
female components from male components differential extraction is used. However,
sometimes separation of male and female components of DNA is not complete as a
result male component also contains female component. In such cases during
amplification of male DNA, female DNA also undergoes amplification and can even
mask the male DNA which in turn makes the autosomal STR analysis difficult. This
masking does not occur while examining Y-STRs. Due to absence of Y STR in
female sample and presence of Y-STR in male sample, the culprit in case of sexual
assault can be linked to the crime. Y-STRs are also useful in non-sexual assault cases
where the evidence materials contain mixed samples of several males. Identification
of all males can be done through Y-STR testing.
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats
6. Mini-STR
Short tandem repeat testing is not successful in case of highly degraded DNA samples
or which are limited in quality or quantity. In such situations only partial STR profile
may be obtained due to drop out of STR alleles. Such partial DNA profiles do not
provide enough information in forensic cases.
Use of mini-STRs is an alternative approach to such cases. For mini STR analysis,
specially designed primers are used which target the mini - STR for amplification.
Mini-STR typing helps in obtaining DNA profile even from highly degraded samples.
7. Characteristics of STRs selected for Human Identification
It is important to have DNA markers which show maximum variation so that they can
be used for human identification. A large number of tetra-nucleotide short tandem
repeats have been discovered which can be used for human identification. Y
chromosome STRs are used for analyzing male-female mixtures from sexual crimes
and in cases where more than one male are involved in the crime.
Following characteristics have been considered for selection of short tandem repeat
loci which have been validated for identification:
i. High discriminating power, usually >0.9, with observed heterozygosity >70%.
ii. The chromosomal locations chosen should be separate to make sure that
closely linked loci are not chosen.
iii. The results should be reproducible and robust when multiplexed with other
markers.
iv. Formation of stutter products (small peaks formed that are several bases
smaller than STR peaks resulting from PCR process when short tandem repeat
loci are copied by a DNA polymerase) should be low.
v. Mutation rate should be low.
vi. For analysis of degraded samples alleles with length in range of 90-500 bps
should be preferred.
vii. The short tandem repeat markers should be chosen from separate chromosome
for forensic DNA typing to avoid any problem with linkage between markers.
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats
Figure 3: Human identification using STR
8. Advantages of STRs over conventional VNTRs
There are several advantages of PCR- based STRs over conventional RFLP technique
of VNTRs (Variable Number of Tandem Repeats):
i. Multiplexing is possible with a narrow allele size range.
ii. Allelic dropouts from preferential amplification of smaller alleles are reduced
due to narrow allele size.
iii. STR can generate small PCR products which help in recovering the
information from degraded DNA samples.
iv. The stutter product formation is reduced as compared to dinucleotide repeats
which help in interpretation of sample mixtures.
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats
9. Applications of STRs
Short tandem repeats (STRs) are small regions in DNA which are analyzed for
various purposes. These are used in forensic testing in crime cases, in missing person
cases and paternity disputes. Besides, STRs are used for following purposes also:
a) Detection of contamination of tissue samples
To understand various normal or abnormal physiological or cellular functions, tissues
are dissected and analyzed. In cases where sample’s identity is disputed, comparison
of STR profile of tissues can be done with reference sample to determine sample’s
identity. This analysis is used to detect sample contamination. The profile of
contaminated sample appears as mixed STR profile. Due to sensitivity of STR
analysis, it can create full profiles from less than 100 pg of DNA. This sensitivity
allows detection of very little quantities of contaminating cells or tissues.
b) Detecting Maternal Cell Contamination and Fetal Aneuploidy
Prenatal samples are contaminated with maternal cells. Before assaying prenatal fetal
sample short tandem repeat analysis is performed to make sure prenatal fetal sample
is not contaminated with maternal cells. This is an important consideration where
maternal DNA can interfere with results. This analysis is also used to detect fetal
blood in maternal blood. STR analysis is used to detect fetal chromosomal
abnormalities like trisomy or aneuploidies. Fetal gender can be determined using
dimorphic Amelogenin locus which distinguishes XX (female) and XY (male)
individuals.
c) Cancer Research
Genetic mutations result in unregulated growth of abnormal cells. This unregulated
growth of cells is known as cancer. Genetic mutations generally occur in tumor
suppressor genes or other proto-oncogenes. To understand all the details of
development and progression of cancer it is necessary to study the associated genetic
changes. The genetic changes can be determined using short tandem repeat loci.
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats
d) Determining Twin Zygosity
To analyze whether the twins are monozygotic or dizygotic, short tandem repeat
analysis is used. STRs are also used for examining the rate of monozygotic twin or
triple births as a result of artificial techniques.
10. Summary
Those regions of DNA which have short repeats (2-7 bps) are known as Short
Tandem Repeats (STR).
STR method is used to compare DNA or particular loci of two or more
samples.
One copy of STR with or without similar repeat numbers is inherited by an
individual from each parent. Variability of number of repeats of STRs is high
among individuals which makes it useful for human identification.
Since, STRs are of small size they can be easily amplified using PCR even
from the degraded samples.
STR loci are highly polymorphic which helps in discrimination between two
DNA profiles.
STRs are of various types: dinucleotides, trinucleotides, tetranucleotides,
pentanucleotides and hexanucleotides.
Short Tandem Repeats are also present on X and Y chromosomes.
Mini-STR primers focus on STR locus only which results in smaller DNA
product. This increases the chances of successful amplification of larger loci.
Due to sensitivity of mini-STR analysis, it can create full profiles from less
than 100 pg of DNA. This sensitivity allows detection of very little quantities
of contaminating cells or tissues.
To detect fetal chromosomal abnormalities like trisomies or aneuploidies STR
analysis is used.
To understand all the details of development and progression of cancer it is
necessary to study the associated genetic changes. The genetic changes can be
determined using short tandem repeat loci.
For informative studies examining the rate of monozygotic, twin or triplet
births as a result of artificial techniques.
FORENSIC SCIENCE PAPER No.13: DNA Forensics
MODULE No.14: Short Tandem Repeats