DNA fingerprinting

DNA Fingerprinting DNA Fingerprinting is also referred to as DNA profiling and DNA typing.

It was first developed as an identification technique in England in 1985.

The original use was to expose the presence of any genetic diseases.

About three years later it became used to identify criminals through the analysis of genetic material and
to settle paternity disputes.

It is still used for those reasons today.

The DNA fingerprinting process is called gel electrophoresis.

It is a process that can sort pieces of DNA according to its size.

It is a way of identifying a specific individual, rather than simply identifying a species or some particular
trait.

DNA Fingerprinting Process

The DNA Fingerprinting Process began in 1985.

Genetic fingerprinting was invented by Sir Alec Jeffreys at the University of Leicester, and is used to
distinguish individuals through DNA.

The process begins by extracting DNA from the cells in a sample of blood,saliva, semen, or other
appropriate fluid or tissue.

An analysis is performed by a cut into the DNA into fragments which are separated into bands.

The bands of DNA are transferred via a technique called Southern blotting.

This is treated with a radioactively-labeled DNA probe which binds to certain and specific DNA
sequences on the membrane.

The excess DNA probe is washed off.

An X-ray film placed next to the nylon membrane detects the radioactive pattern.

This film is then developed to make a visible pattern of bands called DNA fingerprinting.

One of the most modern and widely accepted methods for producing DNA fingerprints in criminal cases,
is that of polymerase chain reaction (PCR).

PCR involves the amplification of specific regions of DNA that are known to be PCR is by far the most
common method for presenting DNA evidence in a forensic context.
Advantages of DNA Fingerprinting :

The most important advantage of DNA fingerprinting is that there is strong similarities shown between
genetic fingerprints of parents and children.

This is a benefit because a child's genetic fingerprint is made up of half the father's genetic information
and half of the mother's information.

This means that the bands of a child's genetic fingerprint will match the bands on both of their parents,
making it possible to establish paternity and maternity tests.

Disadvantages of DNA Fingerprinting:

One of the main problems with the process of DNA fingerprinting is that the sample can be easily ruined.
The tiniest pieces of genetic junk can contaminate DNA samples, causing them to be useless.

Although DNA fingerprinting requires a good sample to work with, this problem can be solved by using
the newer technique called PCR.

PCR can use extremely small samples of DNA and produce a much faster result.

But this also means the DNA samples that PCR uses are even more likely to be contaminated because of
their size, as it is harder to find a small sample with hardly any contamination.

Another limitation of fingerprinting is that the procedure is so complex and hard to read the DNA
patterns, that sometimes the juror finds the evidence almost invisible.

Although DNA Fingerprinting is a highly advanced process, there are still some things that it is unable to
do.

In dogs for example, a fingerprint does not make it possible to determine if the animal is a carrier of a
disease causing allele.

Also, a DNA fingerprint is unable to show a crossbreed in animals.

This is because second or third generation crosses cannot be seen by working backwards in a pedigree.
It may soon become possible to discover the crossbreed of dogs, although right now this is not possible.

Technique

Restriction Fragment Length Polymorphism (RFLP)

Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that
can be detected by the presence of fragments of different lengths after digestion of the DNA samples in

question with specific restriction endonucleases.

RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination.

Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly
locus-specific.

Every strand of DNA has pieces that contain genetic information which informs an organism's
development (exons) and pieces that, apparently, supply no relevant genetic information at all (introns).
Although the introns may seem useless, it has been found that they contain repeated sequences of base
pairs.

These sequences, called Variable Number Tandem Repeats (VNTRs),can contain anywhere from twenty
to one hundred base pairs.

The AFLP technique is based on the selective PCR amplification of restriction fragments from a total
digest of genomic DNA.

The technique involves three steps:

i)restriction of the DNA and ligation of oligonucleotide adapters,

(ii) selective amplification of sets of restriction fragments, and
(iii) gel analysis of the amplified fragments.

How to perform DNA Fingerprinting?

DNA contains two strands which are joined together by the forces between the two nitrogenous bases
on both strands.

We can imagine that DNA is a long ladder with many rungs.

Each rung contains a pair of nitrogenous bases which are distinguished into four different types:
adenine, guanine, cytosine and thymine.

They are in turn symbolized as A, G, C, T respectively.

The sequence of rungs is different for different persons and it is just like paragraphs of alphabets.

Most of the sequence can function to regulate protein synthesis and to regulate cell activities.

The rest of the sequence, in fact, have no specific function but with high variability among people.

So this variability can be used to identify each person.

One common technology used in DNA fingerprinting is the RFLP (restriction fragment length
polymorphism) analysis.

1)The first step is to extract genomic DNA (entire DNA pool of a person) from a person.

This can be done by using one's hairs or blood.

2)The DNA molecules are enclosed in the nucleus of the cells, so the extract of cells needs to be lysed by

breaking the cell membrane and the nucleus envelope to release the DNA molecules.

3)The DNA molecules are now still packed in the form of chromatin by proteins.

4)They are then treated with buffer, enzymes and chloroform so as to destroy the proteins.

5)The pure DNA molecules can then be separated from the destroyed proteins by centrifugation.

6)The extracted DNA molecules are now in a very long chain and need to be cut into many fragments
which are easier to be recognized.

7)The DNA long chains are cut by restriction enzymes.

8)A specific restriction enzyme can cut a specific locus on the DNA chain.

For example,

EcoR1 will only cut the chain at the sequence of GAATTC.

9)The mixture of fragments is then allowed to run in the gel electrophoresis.

The electrophoresis can separate the fragments by electrical charges and a pattern of the fragment can
be obtained eventually.

10)This pattern is different for different persons and hence can be used for distinguishing people.

11)The DNA fragments are then transferred from the gel to a nylon membrane which is called a
Southern Blot.

12)The separated DNA molecules are now still colorless and so it is hard to see them for identification.

13)Probes have to be added to make them visible on a photographic film.

The probe is a small radioactive fragment of DNA which is complementary to and can match with the
sequence of the DNA on the nylon membrane.

14)This matching is called hybridization.

15)The radiation from the probes can cause darkening on the photographic films.
16)This results in the appearance of a pattern of dark bars on the film, which is the DNA fingerprint of a
person.

PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence
as target sites for primer annealing.

The selective amplification is achieved by the use of primers that extend into the restriction fragments,
amplifying only those fragments in which the primer extensions match the nucleotides flanking the
restriction sites.

Using this method, sets of restriction fragments may be visualized by PCR without knowledge of
nucleotide sequence.

The method allows the specific co-amplification of high numbers of restriction fragments.

The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution
of the detection system.

Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels.
The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any
origin or complexity.

Applications

Forensic Uses

Blood or fragments of tissues such as hair and skin cells may be left in the scene of crime.

The DNA fingerprint of these cells from the scene can be revealed and are compared with the DNA
fingerprint obtained from criminal suspects.

If somebody's DNA fingerprint matches with that from the scene this will be a strong evidence showing
that he has committed the crime.

Also in the case of rapes,the semen left in the victims vagina can also be extracted and be used to
reveal the DNA sequence which is later compared with the criminal suspects DNA fingerprints so as to
find out who did the rape.

Paternity

A child can inherit most of the DNA fragment pattern from his parents.

By comparing the DNA fingerprints between the child and the suspect parents, it is possible to identify
who the parent of the child is.