DNA BARCODE
DNA BARCODE
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DNA barcoding is a technique for characterizing species of organisms using a short DNA sequence from a standard and agreed-upon position in the genome.
It differs from molecular phylogeny in that the main goal is not to determine classification but to identify an unknown sample in terms of a known classification.
DNA barcode sequences are very short relative to the entire genome and they can be obtained reasonably quickly and cheaply. The cytochrome c oxidase subunit 1 mitochondrial region (COI) is emerging as the standard barcode region for higher animals.
It is 648 nucleotide base pairs long in most groups, a very short sequence relative to 3 billion base pairs in the human genome.
The ‘barcode’ metaphor is useful though not correct in fine detail.
That is, all the products of one type on a supermarket shelf share exactly the same 11-digit barcode, which is distinct from all other barcodes.
DNA barcodes vary among individuals of the same species, but only to a very minor degree. If the DNA barcode region is effective, the minor variation within species will be much smaller than the differences among species.
APPLICATIONS:
- Identifying plant leaves even when flowers or fruit are not available,
- Identifying insect larvae (which typically have fewer diagnostic characters than adults),
- Identifying the diet of an animal based on stomach contents or faeces, and
- Identifying products in commerce (for example, herbal supplements or wood).
CRITICISM:
DNA Barcoding has met with spirited reaction from scientists, especially systematists, ranging from enthusiastic endorsement to vociferous opposition. Various points of criticism:
- Many stress the fact that DNA Barcoding does not provide reliable information above the species level.
- Some resent what they see as a gross oversimplification of the science of taxonomy.
- Some suggest that recently diverged species might not be distinguishable on the basis of their COI sequences.
DNA PROFILING
KEY POINTS:
v DNA profiling is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles.
v It is also known as DNA testing, DNA typing, or genetic fingerprinting.
v DNA profiles are encrypted sets of numbers that reflect a person’s DNA makeup, which can also be used as the person’s identifier.
v DNA profiling should not be confused with full genome sequencing. It is used in, for example, ‘ parental testing and criminal investigation.
v The DNA profiling technique was first reported in 1984 by Sir Alec Jeffreys and is now the basis of several national DNA databases.
CONCEPT:
Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different to distinguish one individual from another, unless they are monozygotic twins.
DNA profiling uses repetitive (“repeat”) sequences that are highly variable, called Variable Number Tandem Repeats (VNTRs), particularly Short Tandem Repeats (STRs).
VNTR loci are very similar between closely related humans, but so variable that unrelated individuals are extremely unlikely to have the same VNTRs.
Even though we are all unique, most of our DNA is actually identical to other people’s DNA.
However, specific regions vary highly between people, these regions are called polymorphic.
Differences in these variable regions between people are known as polymorphisms.
Each of us inherits a unique combination of polymorphisms from our parents.
DNA polymorphisms can be analysed to give a DNA profile.
The current technique for DNA profiling uses polymorphisms called STRs.
STRs are regions of noncoding DNA that contain repeats of the same nucleotide sequence. For example,
Is an STR where the nucleotide sequence “GATA” is repeated six times. STRs are found at different places or genetic loci in a person’s DNA.
USES OF DNA PROFILING:
v To identify the probable origin of a body fluid sample associated with a crime or crime scene.
v To reveal family relationships
v To identify disaster victims.
What is DNA Fingerprinting?
The chemical structure of everyone’s DNA is the same.
The backbone of the double-stranded DNA is made up of sugar and phosphate. The two strands are held together by four basic units called bases, adenine (A), guanine (G), thiamine (T) and cytosine (C).
The chemical structure of these bases is such that the A of one strand forms a hydrogen bond with the T of another strand and the G of one strand forms hydrogen bond with the C. There is no way A can form hydrogen bond with G or C, and T with G or C.
The only difference between people (or any animal) is in the order of the base pairs.
There are so many millions of base pairs in each person’s DNA that every person has a different sequence.
Using these sequences, every person could be identified solely by the sequence of their base pairs. However, because there are so many millions of base pairs, scientists use a shorter method, based on repeating patterns in DNA.
A class of repetitive DNA such as GATA repeats is differently organized in different individuals.
We may have ten uninterrupted copies of GATA repeats on a particular chromosome in one individual, 30 copies exactly in the same position in another individual, and no copies at all in another individual, and in between any number of copies in other individuals.
The technique by which we can detect the variation in copy number between individuals is called DNA fingerprinting.
This nomenclature was used by Alec Jeffreys to emphasise that DNA pattern of each individual is as unique as are our fingerprints. In fact our DNA fingerprint patterns are much more unique compared to our fingerprints.
A number of non-coding (junk) DNA sequences were isolated by Professor Alec Jeffreys from the flanking region of the globin gene which he used as a probe for DNA fingerprinting. He got it patented and, therefore, it was not available, free of cost, to others.
We in CCMB, therefore, isolated a class of repetitive DNA consisting of GATA repeats from a highly poisonous snake, the Banded krait. We isolated and designated it as Bkm (Banded krait minor satellite) DNA.
We isolated and cloned 545 base pairs of this DNA consisting mostly of GATA repeats We used this by labelling with radioactive and hybridized it with DNA of different individuals, which was cut with appropriate restriction enzymes, size-fractionated on agarose gel by a process called gel electrophoresis, transferred it on a nylon membrane and hybridized with p32 labelled Bkm, exposed this on X-Ray film in the dark and developed it after several days.
One gets a series of bands of different molecular weights containing the GATA repeats. These bands are unique to each individual.
There may be certain bands common in some individuals, but one can never get all the bands exactly the same as in another individual, excepting identical twins who will have identical DNA fingerprinting patterns.
These bands are, therefore, used in forensic investigation. For example, in a family consisting of father, mother and a child, if one conducts DNA fingerprinting test, the pattern in each individual looks very different.
However, when one looks very carefully, every band present in the child is accounted for, either being present in the mother or in the father. There will not be any band present in the child that is not present in either parents.
Normally, maternity is a certainty. If maternity is known, one can establish paternity. If paternity is known maternity can be established. If father and mother are known one can establish the identity of the child.
In the case of brothers and sisters, however, DNA fingerprinting patterns will not be identical. They will be different.
This is because only 23 out of 46 chromosomes are inherited in a child through sperm from the father and 23 chromosomes through the egg from the mother.
Since chromosomes are carriers of DNA, and the bands are generated from the DNA present in the chromosomes, 50% of the bands present in the child are inherited from the mother and 50% from the father.
However, which 50% of the bands are inherited from which parent is a random phenomenon. Therefore, brothers’ and sisters’ DNA fingerprinting patterns will be different.
But if we calculate the co-efficient of band sharing, there will be more than 50% of the bands in common between brothers or brothers and sisters, whereas only 30% of bands would be common between two unrelated individuals.
One can, thus, establish whether the two individuals are brothers or brothers and sisters by this technique. In the case of identical twins, the DNA fingerprinting patterns are identical (Fig).
If we are dealing with a case in which one of the identical twins has actually committed a crime, it would not be possible to identify that individual based on DNA fingerprinting pattern.
These are the limitations of this technique. On the other hand, fingerprints may help in identifying one or the other of the identical twin who has committed the crime because fingerprint patterns of identical twins are slightly different.
Applications of DNA Fingerprinting
DNA fingerprinting is finding extensive use in innumerable areas.
v Crime Investigation: In cases of rape, murder, assassination, theft, etc.
v Authenticity of consumer products: Quite often fake goods are marketed in the name of reputed companies. In order to protect their credibility, many of these top-ranking companies put DNA samples of known DNA sequence underneath each label stuck on each manufactured item. If suspected, these labels can be removed and DNA sequence can be decoded which helps in the authentication of the product.
v Medical diagnosis: In certain diseases such as leukemia, DNA fingerprinting pattern of blood is drastically different compared to the parents of the individual. In such cases, DNA fingerprints can be taken as a signal for identifying the disease.
v Pedigree analysis: Extensively used in horse, cattle and buffalo breeding programmes. There are people who are ready to pay much larger amounts for high pedigree breed known to yield higher quantity of milk, disease- resistant or a much faster runner. It is also being used for determining the pedigree of dogs.
v Seed-stock identification: Used for patenting varieties developed by scientists and released to the farmers. The probability of another scientist developing similar varieties independently having identical DNA pattern is virtually nil. It is also used in molecular breeding of plants.
v Sex-selection in animals: Y chromosome-specific STR markers are used for determining the sex. It is also possible by using FACS to sort Y- bearing sperms from the X-bearing sperms and use them for insemination and production of animals of desired sex.
v Defence records: In US, blood samples of newly recruited soldiers are stored in a freezer. During war when a soldier goes missing, and a mutilated body is discovered and it is physically impossible to identify the body, DNA isolated from the suspect’s blood samples stored earlier is compared with that from the mutilated body to establish identity.
v Wildlife conservation: Extensively used to determine the extent of genetic variation in wild animals and also inbreeding which is very important for the survival of the species. It is also used for determining purity of these species.
v Family matters: Used in establishing paternity or maternity, identity of a missing child and in cases of exchange of babies in maternity wards.
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