DNA Fingerprinting



What is DNA fingerprinting explain:

DNA Fingerprinting 

DNA fingerprinting refers to the process of identifying the characteristics of DNA of individuals. This is done by identifying differences in some specific repetitive DNA sequences. These small stretches of DNA sequences contain DNA stretchs that are repeated by multiple displays. Alec Jeffrey is credited with the development of the DNA fingerprinting technique using the variable number tandem repeats ( VNTRs).


DNA fingerprinting through the VNTR method requires the following five steps:

1): Extraction: DNA is extracted from the small amount of organic material belonging to individuals. This organic material can be in form of blood, semen, hair bulbs, skin cells, etc.

2): Amplication: The polymerase chain reaction ( PCR) is used for creating multiple copies of the extracted DNA.

3): Restriction Digestion:Restriction enzymes are used for cutting the DNA into the required reproducible segments. 


4): Separation: The reproducible segments created in step 3 are separated using gel electrophoresis. 

5): Soythern Blotting: The separated DNA sequences are transferred from the gel onto a nitrocellulose membrane. 
 
DNA   fingerprinting is a technique employed to assist to the identification of individuals on the basis of their respective DNA profiles:

DNA fingerprinting is also known as DNA profiling or DNA typing:

DNA fingerprinting discovered:( Invention of DNA Fingerprinting):

DNA fingerprinting was invented in 1977 by a British geneticist, Sir Alec Jeffrey's at Leicester University, This technique was used for the first time in a regional screen of human DNA to identify the rapist and killer of two girls in Narborough, Leicestershire in 1983 and 1986. Later this technique was refined by developing a more sensitive DNA profiling technique focusing mainly on highly variable minisatellites in the human genome. Now a days, highly automated and sophisticated equipments are employed which can process hundreds of samples in a day.






 

DNA fingerprinting/ DNA profiling/ DNA Barcoding:


■DNA fingerprinting is used to establish the identity of a person or organism based on differences and similarities in DNA.

■DNA profiling was developed in 1984 by Sir Alec Jeffreys of the UK.

■In 1988, this technology was developed in CCMB Hyderabad in India. CCMB stands for Centre for Cell and Molecular Biology.

■In DNA fingerprinting, entire genomics is not used but only some sequence is used to identify a person.


■In DNA, there are some specific sequences known as VNTR or STR- Variable Number of Tandem Repeats and Short Tandem Repeats. Based on this sequence, the DNA is identified.

■Every human being or every organism has VNTR or STR but their number, position and size are different in every organism or human being for analysis of these VNTRs or STR. The similarity and differences in DNA are established.

Technology used in DNA Profiling:

Forensic investigation:


■For the identification of criminals.

■For the identification of a dead body.

■Also used for paternity tests. (Father) famous case N.D. Tiwari's case was resolved through it to identify the kid or baby.

■PCR ( Polymerase chain Reaction): It is used to increase the quality of the DNA ,in case get less DNA .


■ SBT ( Southern Blotting Technology): This helps to identify the specific segments of VNTR and STR of the DNA sample.

■Health care:


■To identify the genetic disorder.

■Pedigree analysis - There is a 25% chance that a particular gene or disease is carried to offspring or the next generation.

■ Electrophoresis: Organic molecules ( like DNA, and RNA protein) can be arranged based on charge and molecular weight .


Some Other DNA profiling Related Technology:


1): Northern Blotting Tech: To identify RNA segments. 

 2): Western Blotting Technology: To identify proteins.

DNA_______RNA_____Protein 

 (SBT)   (NBT)  (WBT)      





■In the conservation of biodiversity:


■To identify the animals and different species.

■To stop the smuggling and hunting of animals. E.g.; To identify dead tigers in other countries. 


Note: DNA fingerprinting is used effectively in forensic science for identifying:
● The biological  father ( in case of paternity disparity)
● The criminals such as murderers and rapists.


Application of DNA Fingerprinting:

1): In forensics, th technique of DNA fingerprinting has been widely used as a remarkable tool solving such complicated cases where the criminal leaves no obvious traces. Under such conditions, even a minute bit of person's body such as skin folicle or a strand of hair can be used to identify the criminal by means of DNA fingerprinting. 


   This technique was used for the first time any where in the world, to identify the person responsible for assassination of late Prime Minister of India, Shri Rajiv Gandhi. Another example of solving a criminal case with  the help of DNA fingerprinting is Tandoor murder case.

2): DNA fingerprinting technique is very well known for its application in paternity testing. This is because the genome of father and mother. Every son/ daughter receives half of the chromosomes from father and half from mother. A close comparision of DNA bands of a child with that of father gives direct evidence of his paternity. This can be easily explained by the following examples,  where son and daughter have been assigned their paternity. The step son, on the other hand, had different DNA bands__India, the first paternity dispute was solved in Tellichery in Kerala using DNA fingerprinting evidence. The evidence was a accepted in the Cout of Law and reported in the annals of Indian judiciary in 1991. Since then several hundred cases have been solved using DNA fingerprinting evidence in various courts. Some argue that DNA evidence is more reliable than an eye witness. 



3): This technique is used to determine the extent of genetic variation in which animals and also study of breeding patterns  i.e., inbreeding or outbreeding. Normally, inbreeding is important for the survival of the species. The technique is also used for determining purity of these species. 


4): DNA fingerprinting is used in determining lineage of human and other animals to ascertain biological evolution. Based on the fact that some parts of the DNA , called " genetic markers", are passed  from generation to generation without any change or modification, the National Geographic Society has launched a project to trace the path of human evolution from their prehistoric roots in Africa. Such study of migration pattern of human beings over long period of time is called Geography. 


5): DNA fingerprinting is used to diagnose hereditary diseases. These diseases include haemophilia, sickle cell anemia, thalassemia, cystic fibrosis, familial Alzheimer's, Huntington's disease, and many others. In certain diseases, DNA fingerprinting is drastically different compared to the parents of the individual. In such cases, this technique can be taken as a signal for identifying the disease. 

6): It is matter of great concern that diseas causing bacteria are developing resistance to antibiotics. Tuberculosis is one example where the pathogenic bacteria is not responding to specific antibiotic.DNA fingerprinting is used to  identify the antibiotic (s) for which  the bacterium is resistant. This technique helps in selection of the right antibiotic for treatment. 


Procedure for DNA Fingerprinting:


(i) DNA molecule are isolated from  a sample of blood, semen, or other body fluid or tissue by using specific techniques such as a high __ speed refrigerated centrifuge. 

(ii) If DNA is in poor condition, or  available only in minute quantities, the particular STRs may be amplified by making many copies of it with the help of using polymerase chain reaction ( PCR).

(iii) DNA molecules are cleaved, i.e.,cut into fragments, at specific sites with the help of a site__ recognizing restriction endonuclease enzyme for the purpose of restriction fragment length analysis. The DNA fragments contain the VNTRs.

(iv) DNA fragments are then sorted out according to their length on an agarose gel by the technique called electrophoresis. The fragments get arranged along the gel according to their length and electric charges.

(v) Double __ stranded DNA is now split into single strands by using alkaline chemicals. 

(vi) The isolated DNA  fragments in the gel are copied into a nylon nitrocellulose sheet placed on the gel.This technique is named Southern blotting after its inventor E.M. Southern. 

(vii) Special DNA probes are prepared in the laboratory, these contain repeated sequences of nucleotides complementary to those on VNTRs. These probes are made radioactive. The radioactive DNA probes bind to the repeat sequence on the nylon sheet. This is called hybridization. An X__ ray film is exposed to the nylon sheet to mark the places where the radioactive DNA probes have bound to the DNA fragment. These places are marked as dark bands when X__ ray film is developed. This process is called autoradiagraphy.

(viii) The dark bands on X_ ray film represent the DNA  fingerprints, or the DNA profiles. 



A summarized account of the procedure of DNA finger printing is diagrammatically represented .



Reliability of DNA Fingerprinting:

The conventional fingerprints commonly used as personal identification were discovered by a Scotish Medical Missionary, Henry Fauld, in 1880.These fingerprints are genetically determined, are unique for an individual, and do not change in one's  life. These fingerprints occur only on the fingers and can be altered by surgery.The DNA fingerprints occur in every cell and cannot be changed by any means.The best identification of an individual would be obtained of his/ her entire genome is subjected to restriction fragments analysis. This is not practicable. Forensic DNA .The DNA regions chosen are ones known to be highly variable from one person to another, and give very accurate results.

Source of DNA for Fingerprinting:

The DNA content of a person is the same for every cell, tissue and organ, the DNA sample for fingerprinting test may be obtained from traces of blood, samen and urine stains on  clothes or other surfaces, saliva residue on cigarette bunds, vaginal swab,tissue bits,  or even a single hair. Besides, DNA can also be isolated from sample that are months or years old.


Principle (Basis) of DNA Fingerprinting:

Genome ( DNA of single set of chromosomes) of a person contain about three billion base pairs.These are arranged in a definite sequences to give an individual his or her identity. Technique of DNA fingerprinting is mainly based on the fact that DNA of  one  individual is a about 90% identical to the DNA of another individual, but there is 10% of the DNA that is different. This 10% of the total DNA  is very important. Scientists use this 10% of the DNA or small number of sequences that are unique among individuals to get a certain probability for a match and distinguish among the individuals. 


   As started earlier, the DNA molecule can be divided into coding and non_ coding sequences. The coding sequence are called exons which carry information required to synthesize proteins. The non__ coding sequences, on the other band, are called introns and do not supply any relevant genetic information. The changes usually occur and accumulate within these non__ coding sequences. These non__ coding sequences of approximately 10 to 100 nucleotides pairs are responsible for variation among individual and can be employed as a tool to create a genetic fingerprint of an individual. Some of these non__ coding sequences contain highly repetitive sequences of base pairs. They are tandomly repeated many times and are found at many place throughout the length of DNA.The number of repeats is very specific in each individual, and it is inherited.It is unlikely that the repeats of one individual are found in the DNA  of another person. 
For example, one individual may have the unit ACA repeated 55 times at one genome locus, 115 times at a second locus, and so on, whereas another individual is likely to have different numbers of repeats at these loci.Some polymorphic genetic loci are usually called simple tandem repeats ( STRs).The identical twins, however, may have the same repeats.


  The tandem repeats make satellite DNA.

For Example,__ ATTICGATICGATTCG__ sequence of base in a DNA  has a very short sequence of _ ATTCG__ repeated times.Such repeated sequences are of two types__ 

(i) Minisatellite: Minisatellites have sequences of approximately 15 to 50 base pairs and repeated tandemly upon thousands of times.There are about 30,000 minisatellite loci in the human genome.

(ii) Microsatellite. The microsatellite have base pairs of less than 10 and repeated usually 10 to 100 times. In human genome, there are about 200,000 such satellite loci.These simple tandem repeats of short sequences are called "Variable number tandem repeats" ( VNTRs). These repeats are inherited from the parents, and are used as genetic markers in a personal identity test.



DNA Fingerprinting:

As stated in the preceding section, 99.9 per cent of base sequence among humans is the same. Assuming human genome as 3× 10 bp, in how many base sequences would there be differences? It is these differences in sequence of DNA which make every individual unique in their phenotypic appearance. If one aims to find out genetic differences between two individuals or among individuals of a population, sequencing the DNA every time would be a daunting and expensive task. Imagine trying to compare two sets of 3× 10⁶ base pairs. DNA fingerprinting is a very quick way to compare DNA sequences of any two individuals. 


  DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called as repetitive DNA, because in these sequences, a sma stretching of DNA is repeated many times. These repetitive DNA are separated from bulk genomic DNADNA as different peaks during density gradient centrifugation. The bulk DNA forms a major peak and the other small peaks are referred to as satellite DNA. 

Depending on base composition ( A: T rich or G: C rich), length of segment, and number of repetitive units, 
The satellite DNA is classified into many categories,such as micro__ satellites, mini __ satellites  etc.
These sequences normally do not code for any proteins, but they form a large portion of human genome, These sequence show high degree of polymorphism and form the basis of DNA fingerprinting. Since DNA from every tissue ( such as blood, hair __ follicle, skin, bone, saliva, sperm etc.), from an individual show the same degree of polymorphism, they become very useful identification tool in forensic applications. Further, as the polymorphisms are inheritable from parents to children,  DNA fingerprinting is the basis of paternity testing, in case of disputes.

Polymorphism in DNA sequence is the basis of genetic mapping of human genome as well as of DNA fingerprinting,it is essential that we understand what DNA polymorphisms means in simple terms.polymorphism (variation at genetic level) arises due to mutations. ( Recall different kind of mutations and their effects that you have already studied and in the preceding sections).New mutations may arise in a  individual either in somatic cells or in the germ cells ( cells that generate in sexually reproducing organisms). If a germ cell mutation does not seriously impair individual's ability to have offspring who can transmit the mutations. It can spread to the other membranes of population ( through sexual reproduction).Allelic sequence variation has traditionally been described as a DNA polymorphism if more than one variant ( allele) at a locus occurs terms, in human population with a frequency greater than 0.01.In simple terms, if an inheritable mutation is observed  in a population at high frequency, it is referred to as DNA Polymorphisms. The probability of such variation to be observed in non__ coding DNA sequence would be higher as mutations in these sequences may not have any immediate effect/ impact in an individual's reproductively ability. These mutations keep on accumulating generation after generation, and form one of the basic of variability/ polymorphism. 

There are a variety of different types of polymorphism ranging from single nucleotide change to vary large scale changes.For evolution and speciation, such polymorphisms play very important role, and you will study these in details at higher classes.


  The technique of DNA Fingerprinting was initially developed by Alec Jeffreys. He used a satellite DNA as probe that shows  very high degree of polymorphism. It was called as Variable Number of Tandem Repeats ( VNTR).The technique, as used earlier, involved Southern blot hybridisation using radiolabelled VNTR as  a probe, It included 


(i) isolation of DNA.

(ii) Digestion of DNA by restriction endonucleases.

(iii) Separation of DNA fragments by electrophoresis, 

(iv) Transferring( blotting) of separated DNA fragments to synthetic  membranes, such as nitrocellulose or nylon,

(v) Hybridisation using labelled VNTR probe, and

(vi) Detection of hybridised DNA fragments by autoradiography. A schematic representation of DNA fingerprinting. 



    Schematic representation of DNA fingerprinting: Few representative chromosomes have been shown to contain different copy number of VNTR.For the sake of understanding different colour schemes have been used to trace the origin of each band in the gel .The two alleles of a chromosome also contain different copy numbers of VNTR. It is clear that the banding pattern of DNA from crime scene matches with individual B, and  not with A.

The VNTR belonged to a class of satellite DNA referred to as mini___ satellite. A small DNA sequence is arranged tandemly in many copy numbers. The copy number varies from chromosome to chromosome in an individual. The number of repeat show very high degree of polymorphism. As a result the size of VNTR varies in size from 0.1 to 20 kb.

Consequently, After hydridisation with VNTR probe, the autoradiogram gives many bands of differing sizes. These bands give a characteristic pattern for an individual DNA .It differs from individual to individual in a population except in the case of monozygotic ( identical) twins. The sensitivity of the technique has been increased by use of polymerase chain reaction .Consequently, DNA from a single cell is enough to perform DNA fingerprinting analysis. 

















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