Starting in the 1980s scientific advances allowed for the use of DNA as a material for the
identification of an individual. The first patent covering the direct use of DNA variation for forensics
was filed by Dr. Jeffrey Glassberg in 1983, based upon work he had done while at Rockefeller
University in 1981. In the United Kingdom, Geneticist Sir Alec Jeffreys[5][6][7][8] independently developed
a DNA profiling process in beginning in late 1984[9] while working in the Department of Genetics at
the University of Leicester.[10]
The process, developed by Jeffreys in conjunction with Peter Gill and Dave Werrett of the Forensic
Science Service (FSS), was first used forensically in the solving of the murder of two teenagers who
had been raped and murdered in Narborough, Leicestershire in 1983 and 1986. In the murder
inquiry, led by Detective David Baker, the DNA contained within blood samples obtained voluntarily
from around 5,000 local men who willingly assisted Leicestershire Constabulary with the
investigation, resulted in the exoneration of a man who had confessed to one of the crimes, and the
subsequent conviction of Colin Pitchfork. Pitchfork, a local bakery employee, had coerced his
coworker Ian Kelly to stand in for him when providing a blood sample—Kelly then used a forged
passport to impersonate Pitchfork. Another coworker reported the deception to the police. Pitchfork
was arrested, and his blood was sent to Jeffrey's lab for processing and profile development.
Pitchfork's profile matched that of DNA left by the murderer which confirmed Pitchfork's presence at
both crime scenes; he pleaded guilty to both murders.[11]
Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is
different that it is possible to distinguish one individual from another, unless they are monozygotic
(identical) twins.[12] DNA profiling uses repetitive sequences that are highly variable,[12] called variable
number tandem repeats (VNTRs), in particular short tandem repeats (STRs), also known
as microsatellites, and minisatellites. VNTR loci are similar between closely related individuals, but
are so variable that unrelated individuals are unlikely to have the same VNTRs.
In India DNA fingerprinting was started by Dr. VK Kashyap and Dr. Lalji Singh. Singh was an Indian
scientist who worked in the field of DNA fingerprinting technology in India, where he was popularly
known as the "Father of Indian DNA fingerprinting".[13]
Profiling processes[edit]
Variations of VNTR allele lengths in 6 individuals.
Alec Jeffreys, a pioneer of DNA profiling.
The process, developed by Glassberg and independently by Jeffreys, begins with a sample of an
individual's DNA (typically called a "reference sample"). Reference samples are usually collected
through a buccal swab. When this is unavailable (for example, when a court order is needed but
unobtainable) other methods may be needed to collect a sample of blood, saliva, semen, vaginal
lubrication, or other fluid or tissue from personal use items (for example, a toothbrush, razor) or from
stored samples (for example, banked sperm or biopsy tissue). Samples obtained from blood
relatives can indicate an individual's profile, as could previous profiled human remains. A reference
sample is then analyzed to create the individual's DNA profile using one of the techniques discussed
below. The DNA profile is then compared against another sample to determine whether there is a
genetic match.
DNA extraction[edit]
When a sample such as blood or saliva is obtained, the DNA is only a small part of what is present
in the sample. Before the DNA can be analyzed, it must be extracted from the cells and purified.
There are many ways this can be accomplished, but all methods follow the same basic procedure.
The cell and nuclear membranes need to be broken up to allow the DNA to be free in solution. Once
the DNA is free, it can be separated from all other cellular components. After the DNA has been
separated in solution, the remaining cellular debris can then be removed from the solution and
discarded, leaving only DNA.[14] The most common methods of DNA extraction include organic
extraction (also called phenol chloroform extraction), Chelex extraction, and solid phase
extraction. Differential extraction is a modified version of extraction in which DNA from two different
types of cells can be separated from each other before being purified from the solution. Each
method of extraction works well in the laboratory, but analysts typically selects their preferred
method based on factors such as the cost, the time involved, the quantity of DNA yielded, and the
quality of DNA yielded.[15] After the DNA is extracted from the sample, it can be analyzed, whether it
is by RFLP analysis or quantification and PCR analysis.
RFLP analysis[edit]
Main article: Restriction fragment length polymorphism
The first methods for finding out genetics used for DNA profiling involved RFLP analysis. DNA is
collected from cells and cut into small pieces using a restriction enzyme (a restriction digest). This
generates DNA fragments of differing sizes as a consequence of variations between DNA
sequences of different individuals. The fragments are then separated on the basis of size using gel
electrophoresis.
The separated fragments are then transferred to a nitrocellulose or nylon filter; this procedure is
called a Southern blot. The DNA fragments within the blot are permanently fixed to the filter, and the
DNA strands are denatured. Radiolabeled probe molecules are then added that are complementary
to sequences in the genome that contain repeat sequences. These repeat sequences tend to vary in
length among different individuals and are called variable number tandem repeat sequences or
VNTRs. The probe molecules hybridize to DNA fragments containing the repeat sequences and
excess probe molecules are washed away. The blot is then exposed to an X-ray film. Fragments of
DNA that have bound to the probe molecules appear as fluoresent bands on the film.
The Southern blot technique requires large amounts of non-degraded sample DNA. Also, Karl
Brown's original technique looked at many minisatellite loci at the same time, increasing the
observed variability, but making it hard to discern individual alleles (and thereby precluding paternity
testing). These early techniques have been supplanted by PCR-based assays.
Polymerase chain reaction (PCR) analysis[edit]
Main article: Polymerase chain reaction
Developed by Kary Mullis in 1983, a process was reported by which specific portions of the sample
DNA can be amplified almost indefinitely (Saiki et al. 1985, 1985) The process, polymerase chain
reaction (PCR), mimics the biological process of DNA replication, but confines it to specific DNA
sequences of interest. With the invention of the PCR technique, DNA profiling took huge strides
forward in both discriminating power and the ability to recover information from very small (or
degraded) starting samples.
PCR greatly amplifies the amounts of a specific region of DNA. In the PCR process, the DNA
sample is denatured into the separate individual polynucleotide strands through heating.
Two oligonucleotide DNA primers are used to hybridize to two corresponding nearby sites on
opposite DNA strands in such a fashion that the normal enzymatic extension of the active terminal of
each primer (that is, the 3’ end) leads toward the other primer. PCR uses replication enzymes that
are tolerant of high temperatures, such as the thermostable Taq polymerase. In this fashion, two
new copies of the sequence of interest are generated. Repeated denaturation, hybridization, and
extension in this fashion produce an exponentially growing number of copies of the DNA of interest.
Instruments that perform thermal cycling are readily available from commercial sources. This
process can produce a million-fold or greater amplification of the desired region in 2 hours or less.
Early assays such as the HLA-DQ alpha reverse dot blot strips grew to be very popular owing to
their ease of use, and the speed with which a result could be obtained. However, they were not as
discriminating as RFLP analysis. It was also difficult to determine a DNA profile for mixed samples,
such as a vaginal swab from a sexual assault victim.
However, the PCR method was readily adaptable for analyzing VNTR, in particular STR loci. In
recent years, research in human DNA quantitation has focused on new "real-time" quantitative PCR
(qPCR) techniques. Quantitative PCR methods enable automated, precise, and high-throughput
measurements. Inter-laboratory studies have demonstrated the importance of human DNA
quantitation on achieving reliable interpretation of STR typing and obtaining consistent results across
laboratories.
STR analysis[edit]
Main article: Short tandem repeats
The system of DNA profiling used today is based on polymerase chain reaction (PCR) and uses
simple sequences[16] or short tandem repeats (STR). This method uses highly polymorphic regions
that have short repeated sequences of DNA (the most common is 4 bases repeated, but there are
other lengths in use, including 3 and 5 bases). Because unrelated people almost certainly have
different numbers of repeat units, STRs can be used to discriminate between unrelated individuals.
These STR loci (locations on a chromosome) are targeted with sequence-specific primers and
amplified using PCR. The DNA fragments that result are then separated and detected
using electrophoresis. There are two common methods of separation and detection, capillary
electrophoresis (CE) and gel electrophoresis.
Each STR is polymorphic, but the number of alleles is very small. Typically each STR allele will be
shared by around 5–20% of individuals. The power of STR analysis derives from inspecting multiple
STR loci simultaneously. The pattern of alleles can identify an individual quite accurately. Thus STR
analysis provides an excellent identification tool. The more STR regions that are tested in an
individual the more discriminating the test becomes.
From country to country, different STR-based DNA-profiling systems are in use. In North America,
systems that amplify the CODIS 20[17] core loci are almost universal, whereas in the United Kingdom
the DNA-17 17 loci system (which is compatible with The National DNA Database) is in use, and
Australia uses 18 core markers.[18] Whichever system is used, many of the STR regions used are the
same. These DNA-profiling systems are based on multiplex reactions, whereby many STR regions
will be tested at the same time.
The true power of STR analysis is in its statistical power of discrimination. Because the 20 loci that
are currently used for discrimination in CODIS are independently assorted (having a certain number
of repeats at one locus does not change the likelihood of having any number of repeats at any other
locus), the product rule for probabilities can be applied. This means that, if someone has the DNA
type of ABC, where the three loci were independent, then the probability of that individual having that
DNA type is the probability of having type A times the probability of having type B times the
probability of having type C. This has resulted in the ability to generate match probabilities of 1 in a
quintillion (1x1018) or more. However, DNA database searches showed much more frequent than
expected false DNA profile matches.[19] Moreover, since there are about 12 million monozygotic
twins on Earth, the theoretical probability is not accurate.
In practice, the risk of contaminated-matching is much greater than matching a distant relative, such
as contamination of a sample from nearby objects, or from left-over cells transferred from a prior
test. The risk is greater for matching the most common person in the samples: Everything collected
from, or in contact with, a victim is a major source of contamination for any other samples brought
into a lab. For that reason, multiple control-samples are typically tested in order to ensure that they
stayed clean, when prepared during the same period as the actual test samples. Unexpected
matches (or variations) in several control-samples indicates a high probability of contamination for
the actual test samples. In a relationship test, the full DNA profiles should differ (except for twins), to
prove that a person was not actually matched as being related to their own DNA in another sample.
AFLP[edit]
Main article: Amplified fragment length polymorphism
Another technique, AFLP, or amplified fragment length polymorphism was also put into practice
during the early 1990s. This technique was also faster than RFLP analysis and used PCR to amplify
DNA samples. It relied on variable number tandem repeat (VNTR) polymorphisms to distinguish
various alleles, which were separated on a polyacrylamide gel using an allelic ladder (as opposed to
a molecular weight ladder). Bands could be visualized by silver staining the gel. One popular focus
for fingerprinting was the D1S80 locus. As with all PCR based methods, highly degraded DNA or
very small amounts of DNA may cause allelic dropout (causing a mistake in thinking a heterozygote
is a homozygote) or other stochastic effects. In addition, because the analysis is done on a gel, very
high number repeats may bunch together at the top of the gel, making it difficult to resolve. AmpFLP
analysis can be highly automated, and allows for easy creation of phylogenetic trees based on
comparing individual samples of DNA. Due to its relatively low cost and ease of set-up and
operation, AmpFLP remains popular in lower income countries.
DNA family relationship analysis[edit]
1: A cell sample is taken – usually a cheek swab or blood test 2: DNA is extracted from sample 3: Cleavage of
DNA by restriction enzyme – the DNA is broken into small fragments 4: Small fragments are amplified by the
polymerase chain reaction – results in many more fragments 5: DNA fragments are separated by
electrophoresis 6: The fragments are transferred to an agar plate 7: On the agar plate specific DNA fragments
are bound to a radioactive DNA probe 8: The agar plate is washed free of excess probe 9: An x-ray film is used
to detect a radioactive pattern 10: The DNA is compared to other DNA samples
Using PCR technology, DNA analysis is widely applied to determine genetic family relationships
such as paternity, maternity, siblingship and other kinships.
During conception, the father's sperm cell and the mother's egg cell, each containing half the amount
of DNA found in other body cells, meet and fuse to form a fertilized egg, called a zygote. The zygote
contains a complete set of DNA molecules, a unique combination of DNA from both parents. This
zygote divides and multiplies into an embryo and later, a full human being.
At each stage of development, all the cells forming the body contain the same DNA—half from the
father and half from the mother. This fact allows the relationship testing to use all types of all
samples including loose cells from the cheeks collected using buccal swabs, blood or other types of
samples.
There are predictable inheritance patterns at certain locations (called loci) in the human genome,
which have been found to be useful in determining identity and biological relationships. These loci
contain specific DNA markers that scientists use to identify individuals. In a routine DNA paternity
test, the markers used are short tandem repeats (STRs), short pieces of DNA that occur in highly
differential repeat patterns among individuals.
Each person's DNA contains two copies of these markers—one copy inherited from the father and
one from the mother. Within a population, the markers at each person's DNA location could differ in
length and sometimes sequence, depending on the markers inherited from the parents.
The combination of marker sizes found in each person makes up his/her unique genetic profile.
When determining the relationship between two individuals, their genetic profiles are compared to
see if they share the same inheritance patterns at a statistically conclusive rate.
For example, the following sample report from this commercial DNA paternity testing laboratory
Universal Genetics signifies how relatedness between parents and child is identified on those
special markers:
DNA
Mother Child Alleged father
marker
D21S11 28, 30 28, 31 29, 31
D7S820 9, 10 10, 11 11, 12
TH01 14, 15 14, 16 15, 16
D13S317 7, 8 7, 9 8, 9
14,
D19S433 14, 15 15, 17
16.2
The partial results indicate that the child and the alleged father's DNA match among these five
markers. The complete test results show this correlation on 16 markers between the child and the
tested man to enable a conclusion to be drawn as to whether or not the man is the biological father.
Each marker is assigned with a Paternity Index (PI), which is a statistical measure of how powerfully
a match at a particular marker indicates paternity. The PI of each marker is multiplied with each
other to generate the Combined Paternity Index (CPI), which indicates the overall probability of an
individual being the biological father of the tested child relative to a randomly selected man from the
entire population of the same race. The CPI is then converted into a Probability of Paternity showing
the degree of relatedness between the alleged father and child.
The DNA test report in other family relationship tests, such as grandparentage and siblingship tests,
is similar to a paternity test report. Instead of the Combined Paternity Index, a different value, such
as a Siblingship Index, is reported.
The report shows the genetic profiles of each tested person. If there are markers shared among the
tested individuals, the probability of biological relationship is calculated to determine how likely the
tested individuals share the same markers due to a blood relationship.
Y-chromosome analysis[edit]
Recent innovations have included the creation of primers targeting polymorphic regions on the Y-
chromosome (Y-STR), which allows resolution of a mixed DNA sample from a male and female or
cases in which a differential extraction is not possible. Y-chromosomes are paternally inherited, so
Y-STR analysis can help in the identification of paternally related males. Y-STR analysis was
performed in the Sally Hemings controversy to determine if Thomas Jefferson had sired a son with
one of his slaves. The analysis of the Y-chromosome yields weaker results than autosomal
chromosome analysis. The Y male sex-determining chromosome, as it is inherited only by males
from their fathers, is almost identical along the patrilineal line. This leads to a less precise analysis
than if autosomal chromosomes were testing, because of the random matching that occurs between
pairs of chromosomes as zygotes are being made.[20]
Mitochondrial analysis[edit]
Main article: Mitochondrial DNA
For highly degraded samples, it is sometimes impossible to get a complete profile of the 13 CODIS
STRs. In these situations, mitochondrial DNA (mtDNA) is sometimes typed due to there being many
copies of mtDNA in a cell, while there may only be 1–2 copies of the nuclear DNA. Forensic
scientists amplify the HV1 and HV2 regions of the mtDNA, and then sequence each region and
compare single-nucleotide differences to a reference. Because mtDNA is maternally inherited,
directly linked maternal relatives can be used as match references, such as one's maternal
grandmother's daughter's son. In general, a difference of two or more nucleotides is considered to
be an exclusion. Heteroplasmy and poly-C differences may throw off straight sequence
comparisons, so some expertise on the part of the analyst is required. mtDNA is useful in
determining clear identities, such as those of missing people when a maternally linked relative can
be found. mtDNA testing was used in determining that Anna Anderson was not the Russian princess
she had claimed to be, Anastasia Romanov.
