DNA FORENSICS
Andy Cheng, Mike Garofalo, Jon Keung, Bo Li, Jenny Nguyen, Jeff Wharton
Introduction:
DNA forensics?
DNA Forensics a form of forensics that harness the minute differences
that exist between individual’s genetic material in order to distinguish, identify,
and match subjects.
The differences (polymorphisms) between the genetic materials can be
examined and used in objective ways in fields such as archeology, crime
forensics, identification of species, and even agricultural monitoring. The field
was greatly advanced by the advent of DNA-based identification techniques.
There exist several powerful techniques, each relies on a specific characteristic
of DNA sequence, but usually involve laboratory examination of the sequence
or length of unique regions of DNA.
The techniques that are commonly used are Restriction Length
Polymorphisms (RFLPs), Short Tandem Repeats (STRs), and analysis of
mitochondrial DNA and the Y-chromosome. Each of the above approach carries
its own set of advantages and limitations.
DNA based forensics relies on differences between DNA samples.
These differences can be inherited or obtained from random mutation events,
but this only occurs rarely. Polymorphisms without a selectional pressure
occurs at a fixed rate. Forensics utilizes these frequencies to calculate the
probability of an individual having a certain set of polymorphisms.
As DNA-based forensic techniques improve and become more efficient,
the costs and implications of using these techniques must be considered.
Figure 1. Electrophoresis gel of RFLPs analysis.
The different bands are the different lengthed fragment
generated after digestion with restriction enzyme.
Since there are a number of polymorphisms in the genome it happens
that some of these variations would occur at the recognition sequences for
restriction enzymes. If an enzyme would normal recognize a sequence such as
GAATTC (for EcoR1), it would inhibit the enzyme’s ability to cleave the DNA, or
conversely a non-cleavage sequence could be changed to a recognition
sequence, causing the enzyme to cut the DNA. These different cleavage sites
lead to differences in the length of the DNA segments after digestion. (An
example of a RFLPs gel is seen in Figure 1). When the digested DNA is run
with gel electrophoresis, there can be a detectible difference in the bands from
two individuals. This information is useful in criminal investigations as well as
paternity tests.
BamH1
GGATCC
Hae III
GGCC
Pst I
CTGCAG
Hinf I
GANTC
Table 1. Restriction enzyme and the
core recognition sequene
associated. Any change in the core
sequen will result in large decrease
in efficiency of enzyme.
Figure 2. Outline of RFLPs procedure. DNA
is first extracted and digested by a set of
restriction enzymes. Fragments are
separated by electrophoresis and Southern
blott performed as methods of detection.
Results can be compared to other samples
that have also undergone the same
reactions, with the same mixture of
restriction enzymes.
Detection can also be done via
electrophoresis (not shown). A large amount
of sample needed.
Restriction Length Polymorphisms (RFLPs).
Restriction enzymes cleave double stranded DNA by hydrolyzing the
backbone between the deoxyribose sugar and the phosphate at the 5’
phosphate and the 3’-OH. These enzymes bind, loosely at first, to the DNA and
move along it until it comes in contact with the recognition sequence. The
tighter binding with the recognition sequence then leads to a conformational
change that allows the enzyme to cleave the DNA.
Sequence
An increase in the research of STR has led to the discovery of a number of
STR on the Y-chromosome. These “Y-STR” are important because they can help track
lineage better than autosomal STR due to the fact that the Y-chromosome is solely
paternally inherited.
While both RFLP and STR are very common and very effective methods of
DNA forensics, RFLP is more widespread and preferable to STR. This is due to the fact
that RFLP is more sensitive to mutations and can therefore give more conclusive
results. A difference of one or two repeats is very difficult to see on a gel, while one
mutation on a genome at a recognition site for an enzyme will yield a very different band
on the gel.
Cost and Efficiency
DNA Forensic Techniques
RFLPs are differences in the length of DNA fragments after digestion
with restriction endonucleases. These differences in length are caused by
variations, or polymorphisms in the DNA sequences in the human genome.
While most of the human genome is identical from individual to individual, about
3 million single nucleotide polymorphisms (SNPs) exist, the genome itself being
approximately 3 billion base pairs.
Enzyme
Short Tandem Repeat Analysis (STRs)
Another method of DNA identification makes use of DNA repeats. There are
two classes of repeats Variable Number Tandem Repeats (VNTRs) with a core
sequence of 20-9 bps and Short tandem repeats (STRs) with a core sequence of 2-5
bps. STRs are short segments of DNA that are repeated a number of times in a
sequence. Across the population, different individuals have different numbers of these
repeats. The lengths of DNA of STR loci are usually smaller than RFLP fragments and
hence are better for using on damaged DNA. When a DNA polymerase encounters a
repeated sequence it has a tendency to stutter or slip a little, leading to increases and
decreases in the number of repeats, which is a source of polymorphisms. By
determining the sequences that flank the STR, and amplifying them via PCR
(polymerase chain reaction), the differences in length can be detected via gel
electrophoresis. An individual with an increased number of repeats will have a longer
segment of DNA, and therefore a higher band on the gel.
In addition to running size exclusion gel electrophoresis, the STR can be
identified with sequencing. While the price of DNA sequencing has gone down
dramatically, it is still more expensive than running a gel, and it also takes longer.
Figure 3. Sequencing of STRs.
Since STRs are small in size,
sequencing can be applied to STR
analysis as well as separation by
electrophoresis.
Cost:
- Cost of processing of DNA is approximately $50.00 per sample ~ $2.5 million/year/state
(Michigan State’s House Fiscal Agency).
- Private samples can be sent in and processed at company called Genex Diagnostics™ for
approximately $180.00 per sample
- RFLP is the most common – techniques have become much cheaper lately – hybridization
solutions composed of cost-effective reagents (7% SDS, 10% PEG, and phosphate buffer)
Efficiency:
- Efficiency of DNA forensics analysis continuously increasing and is now much easier.
- 90% of analys is done by computers with little room for human error
- With longer autoradiographic exposures, as little as 20-100 ng of genomic DNA is sufficient for
analysis
Reliability:
-Very reliable – DNA comparisons nearly foolproof
-Reliability increases with more probes used and matched between samples
-RFLP is more reliable because tests can be much more variable
CODIS
The Federal Bureau of Investigation (FBI) uses a system that combines
computerized matching algorithms and forensic science to solve violent crimes. The
system is called the COmbined DNA Index System or CODIS. Since it’s inception as a
small pilot program in 1990 the CODIS system has grown to include over 3 million
samples and is now relied upon to solve crimes nationwide. It has become an invaluable
tool, assisting in more than 33 thousand investigations. CODIS is divided into three tiers
customized to the local, state, and national level. The DNA Identification Act of 1994
established CODIS as the national DNA database for logging the DNA collected from
violent crimes. All states are required by law to participate in the program.
The CODIS system contains two main databases referred to as the Forensic and
Offender Index.
DNA profile from sexual and
violent criminals automatically
entered into database.
Offender
Index
DNA samples from crime
scenes collected and entered
into database.
Match!
Forensic
Index
Victim / Perpetuator / Accomplice Identified
The Offender Index contains
DNA samples from individuals
convicted of sexual and violent
crimes. The Forensic Index
contains DNA samples from
the crime scenes of unsolved
crimes. Matches between the
Forensic and Offender Index
can give officers leads, identify
the perpetuator, and make a
stronger case against serial
offenders.
Creating the DNA Profile
A DNA Profile is generated by analyzing the number of tetranucleotide single
Picture shows sequencing result
tandem repeats (STRs) in 13 loci of the human genome. The odds that two persons
of a CCAACC repeat region.
would contain the same number of repeats at each locus are approximately one in one
Limitations of RFLP
billion. The STR loci are isolated and amplified using PCR. Exhaustive studies have
shown an absence of false positives and negatives while using the system (Moretti et al
A large sample size is required to run a RFLP gel. The technique
2001). However, the system does have limitations when DNA evidence from two or more
detects the length of cut DNA. DNA that is heavily damaged cannot be used. If
persons becomes intermixed. A recent study showed that a three person intermixture
only a small sample size is available, RFLP may still be usable if coupled with
could be construed as only 2 persons 3% of the time, whereas a 4 person genetic
PCR. PCR can amplify a small sample into a larger one. However,
mixture could be construed as a 2 or 3 person mixture 70% of the time (Paoletti et al
contamination of sample may cause amplification other than the desired DNA.
2005). Contamination and sample degradation can also inhibit the ability to make a
genetic profile.
Despite these limitations the CODIS system has been used to aide and solve
Hammer, Michael, Alan J. Redd. “Forensic Applications of Y-Chromosome STRs and SNPs” Deparment of Justice. Jan 2006 http://72.14.203.104/search?q=cache:hBWiApYKTT4J:www.ncjrs.gov/pdffiles1/nij/grants/211979.pdf+Y-chromosome+forensics%22&hl=en&gl=us&ct=clnk&cd=1
“Blackett Family DNA Activity 2” The Biology Project: University of Arizona. Oct 2000. http://www.biology.arizona.edu/Human_Bio/activities/blackett2/str_description.html
thousands of crimes. The ability to identify a biological sample from a crime scene to
Philpot , Jen “DNA FINGERPRINTING IN THE STANDARDIZATION OF HERBS AND NUTRACEUTICALS” Bioteach http://www.bioteach.ubc.ca/MolecularBiology/DNAfingerprintherbs/rflp.gif
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known convicted criminals has proven to be an effective tool of law enforcement.
Simmer, Megan, Dave Secko “Restriction Endonucleases: Molecular Scissors for Specifically Cutting DNA” Bioteach http://bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/
Moretti, T., Baumstark, A., Degenbaugh, D., Keys, K., Smerick J. (2001) Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples. Journal of Forensic Science. 46 (3): 647-60.
Paoletti, D., Doom, T., Krane, C., Yamer, M, Krane, D. (2005) Empirical analysis of the STR profiles resulting from conceptual mixtures. Journal of Forensic Science. 50 (6): 1361-6.