DNA Fingerprinting

DNA FINGERPRINTING

(DNA PROFILING)

Table Of Content1. Introduction of DNA Fingerprinting.

2. Stages involved.

3. Principle of DNA Fingerprinting.

4. Importance of DNA Fingerprinting.

5. Diagram.

6. DNA Profiling Process.

7. Applications of DNA Fingerprinting.

8. Advantages and Disadvantages of DNA Fingerprinting.

9. Uses of DNA Fingerprinting.

10. Benefits and limits of DNA Fingerprinting.

11. DNA Fingerprinting in plants.

12. Uses of DNA Fingerprinting in Forensic Science

13. Centers for DNA Fingerprinting.

14. Conclusion.

Introduction of DNA Fingerprinting:

• 1980 - American researchers

discovered non-coding regions of

DNA

• 1984 - Professor Alec Jeffreys

developed the process of DNA

profiling

• 1987 - First conviction based on

DNA evidence

• A process or technique of analysis

• revealing unique patterns of an individual’s DNA

• involving non-coding regions

Dna profiling

DNA FINGERPRINTING( DNA PROFILING )

STAGES INVOLVED:

•Cells broken down to release DNA

•DNA strands cut into fragments

•Fragments separated

•Pattern of fragments analysed

1. DNA EXTRACTION

DNA FINGERPRINTING

2. DNA CUTTING 3. FRAGMENT SEPARATION

The samples containing the fragments are pipetted into individual wells in a gel

DNA FINGERPRINTING

3. ELECTROPHORESIS

•Fragments separated by length

•DNA (negatively charged)

•Moves towards +ve terminal

•Shorter fragments move faster

DNA FINGERPRINTING

4.DNA TRANSFER

•DNA split into single strands using alkaline solution•DNA fragments transferred from gel to filter paper or nylon membrane• (This is called Southern blotting)•Gel, with filter paper attached, is removed & separated

Radioactive probe in solution binds to DNA

Revealing a pattern of bands

X-ray film

5. Analysis


Principle of DNA Fingerprinting:

•Base pairing of AT (AU) & GC is the BASIC PRINCIPLE of this procedure.

• Specificity of enzyme activity is the second CRUCIAL principle to understanding DNA

fingerprinting. This refers to the cutting of DNA by specific RESTRICTION ENZYMES at

UNIQUE palindrome sequences.

•The recognition that a CHANGE in a SINGLE BASE PAIR (mutation) can either

MAKE a restriction enzyme-site where one did not exist previously or it can REMOVE or

ELIMINATE a restriction enzyme site from a gene. An analogy would be to "mutate" your

phone number by one letter; callers would get a different person.

Importance of DNA Fingerprinting:

1. Lineage

DNA fingerprinting can be used to figure out if two people are related which aids

tremendulously in maternity or paternity tests to show that a person is a mother or father to

a specific child. DNA fingerprinting can also be used to trace heritage back for

generations. This means that not only can you figure out who your mother and father are,

but also siblings, aunts, uncles, grandparents, great grandparents, and even further back.

DNA fingerprinting has been able to show relations to people today with bone remnants of

people that have been dead for centuries, when bones have been preserved.


2. Law Enforcement

DNA fingerprinting has aided in law enforcement since its discovery. Forensics teams can

analyze DNA found at a crime scene, whether it be blood, hair, semen, or skin particles,

and compare it with DNA samples found in a DNA database to find out who committed a

crime. DNA fingerprinting can also be used to identify a victim even if the person is

disfigured beyond recognition and dental records cannot be established. DNA

fingerprinting has been used to prove suspects guilty of a crime and has set innocent people

free when earlier evidence has proven them guilty. DNA fingerprinting is not perfect and

there is still much controversy over whether it can be used in a court of law, but regardless,

it remains an essential part of our criminal justice system.


3. Personal Identification

DNA fingerprinting has also been thought of as a future method of identification. The way it

would work is that a sample of DNA could be analyzed on spot and compared to a database

of specific DNA belonging to authorized personnel. While DNA is the ultimate bar code, it

would be too expensive and impractical to use DNA fingerprinting for personal

identification while other means of identification such as picture ID and social security

numbers (although both can be faked) are available.


DNA Profiling Process:

•Variable Number Of Tandem Repeats(VNTRs)

• Amplified Length Polymorphisms (AFLPs)

•Restriction Fragment Length Polymorphism (RFLP)


1.Variable Number Of Tandem Repeats(VNTRs)

•Most of the DNA in a chromosome does not code for a gene •These regions contain sequences that repeat from 20-100 times (ex- GTCAGTCAGTCAGTCA) •There are several known variations of the Short Tandem Repeats (STR) in humans

•Ex.- HUMTH01 (repeating AATG) has seven different variations


•Scientists can look at 4-6

different STR’s in the same

person

•The more STR’s identified the

higher the probability of a match

•VNTR is more commonly used

than RFLP

VNTR

Advantages of VNTR

• Less complex patterns in gel

•STR’s are less likely to degrade

Less than 400 base pairs

In the middle of the chromosome

• Can be used on stains that old or have been exposed to

decomposition

( DNA PROFILING )

( DNA PROFILING )

2. Amplified Length Polymorphisms (AFLPs)

AFLP definition: Any difference between corresponding DNA fragments from two organisms A and B that is detected by the amplified restriction length polymorphism technique (Kahl, 2001).

•Initially, DNA is extracted as for RFLP and then cut with two different restriction enzymes to produce well defined restriction fragments with sticky ends. •Synthetic double stranded linkers of approximately 18 to 20 bp with matching sticky ends are ligated on all the restriction fragments.

•Ligated fragments are subsequently amplified in PCR with 18 to 20 nucleotide length primers recognizing linkers in each end of the fragments

•A primer used for amplification in AFLP is normally labeled with P33 or some nonradioactive labeling system.

•The mixture of amplified fragments are separated according to size in a Polyacrylamide gel and visualized by means of autoradiography or other procedures for development of non radioactive systems.

•Alternatively, automated sequencing machines can be used to read the sequence of amplification products directly from the Polyacrylamide gel if primers have been labeled with special fluorescent dyes.

•Also, silver staining of the Polyacrylamide gel after electrophoresis can be used to stain all amplified DNA fragments in the gel

•To avoid that all restriction fragments from the genome amplify in the same PCR, which will produce a smear because of too many DNA fragments, a two step amplification procedure is used.

•Firstly, part of the total number of restriction fragments are amplified in the "preamplification" with primers containing one extra "selective" nucleotide on their 3' end.

•This selective nucleotide will allow amplification only of restriction fragments with a matching nucleotide next to the linker.

•With one selective nucleotide on both primers only 1/16th of all restriction fragments in the mixture will amplify during the preamplification.

•During the subsequent "selective" amplification, additionally, one or two selective nucleotides on each primer will further reduce the number of fragments amplified.

•A good AFLP amplification will show 90 to 100 different fragments in one analysis.

The sequence of adapters and primers used in this study is shown below:

MseI adaptor: 5-GACGA TGA GTCC TGAG-3 3-T ACT CGG ACTCAT-5

EcoRI adaptor: 5-biotin-CTCGTA GAC TGCG TACC-3 3-CTG ACGC ATGGTTAA-5 MseI + 1 primer : 5 - GATGAGTCCTGAGTAAA MseI + 3 primers : 5 - GATGAGTCCTGAGTAAAAC EcoRI + 1 primer : 5 - GACTGAGTACCAATTCA EcoRI + 3 primers : 5 – GACTGCGTACCAATTCACG

Selective amplification is then carried out by PCR using preamplified DNA fragments with EcoRI and MseI primer with three-selected nucleotide each. The final PCR products are separated and detected by

Polyacrylamide gel electrophoresis.

3.Restriction Fragment Length Polymorphism (RFLP)

Steps of making an RFLP:

1. Add a restriction enzyme to cut the DNA into fragments - Exact number and

size of fragments produced varies from person to person

2. Fragments are separated by electrophoresis

3. The smaller fragments travel further than the

large fragments

4. This creates a DNA Fingerprint


( DNA PROFILING )

( DNA PROFILING )


Applications of DNA Fingerprinting:

1. Paternity and Maternity

Because a person inherits his or her VNTRs from his or her parents, VNTR patterns canbe used to establish paternity and maternity. The patterns are so specific that a parental VNTR pattern can be reconstructed even if only the children VNTR patterns are known (the more children produced, the more reliable the reconstruction). Parent-child VNTR pattern analysis has been used to solve standard father-identification cases as well as more complicated cases of confirming legal nationality and, in instances of adoption, biological parenthood.


2. Criminal Identification and Forensics

DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to determine guilt or innocence. VNTR patterns are also useful in establishing the identity of a homicide victim, either from DNA found as evidence or from the body itself.

3. Personal Identification

The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen.

Advantages And Disadvantages of DNA Fingerprinting:

Advantages:

•DNA profiling is an ideal method for confirming an identity with absolute certainty.

•It’s easy and painless to obtain a specimen for testing.

•A thorough, scientific test can be conducted in as little as 48 hours.

•DNA testing is affordable and reliable.


Disadvantages:

To be conclusive, a DNA test should be run on multiple samples, at least twice. DNA People Diagnostics collects four samples and the lab runs every test twice to avoid false readings. In fact, your samples are sent to the only lab in the country to actually run each test twice through two separate DNA analyzers, virtually eliminating the chance for error. The lab also tests up to 29 markers to produce the highest possible probability indicators. Most labs will only test 10 markers, greatly increasing the need for additional testing. While most labs charge patients when this occurs, we do not charge when additional testing is necessary. Misuse of results can lead to privacy concerns. DNA People Diagnostics guarantees complete confidentiality. Personal information will not be released to anyone other than the tested parties and their named representatives without a written order.


Uses Of DNA Fingerprinting:

Medical researchers, forensic biologists, genealogists and anthropologists all use DNA fingerprinting to identify individuals or to determine the relation between individuals or groups of individuals. Information gained from DNA fingerprinting can answer questions concerning the structure and migration of human populations, establish paternity and place a suspect at the scene of a crime.

( DNA PROFILING )

Benefits and limits of DNA Fingerprinting:

Benefits of DNA Fingerprinting: The most important benefit 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.

The above picture shows how a paternity test is used to match a child with their biological father. It shows that the child was compared with his alleged father, and the test on the right shows the child's DNA matches the father's DNA. This means that the father on the right is his biological father, while the one on the left is not.

Limitations of DNA Fingerprinting:

An uncontaminated DNA blood sample taken from a crime scene.

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.

( DNA PROFILING )

DNA Fingerprinting In Plants:

The basic methodology of DNA profiling in plants involve first the extraction of DNA from plant cells, quantification and quality assessment of extract. The further steps are of two types, 1) PCR based. - RAPD, ISSR, SSR 2) Non PCR based. – RFLP.

The in the PCR based techniques diluted DNA is mixed with a master mix comprising the PCR buffer, DNTPS, primer, water and the Taq polymerase enzyme in a PCR eppendorf tube .The mixture is loaded into the PCR. The PCR is pre-programmed for appropriate number of cycles and temperature variations depending on the technique. After required cycles, the samples are subjected to electrophoresis, either AGE or PAGE, depending on the technique. The staining is done for revealing the banding pattern.

•The more STRs that are compared from each DNA sample, the less likely the chance will be that they have arisen from the same source. The likelihood of any two specimens being identical is calculated using the 'product rule'. Here, the probability of the frequency of occurrence of each STR in a population is multiplied by that of the other STRs separated from the DNA sample.

•During the O.J. Simpson trial in 1995, for instance, O.J.’s guilt was suggested by STR tests carried out on his blood and blood from the crime scene, which revealed the probability of his being innocent was 1 in 240000. Further RFLP tests narrowed this down to 1 in 57 billion. The main reason he wasn’t convicted was due to the seed of doubt the defense showed in the minds of the jurors about possible interference with the evidence by the FBI.

•In addition to its use in providing evidence to incriminate suspects, DNA profiling can also be of use in exonerating suspects accused of crimes. About 25% of violent crime cases in the U.S. since 1989 have resulted in the exoneration of suspects because of DNA profiling procedures. By 1996, over 108 post-conviction exonerations had in fact occurred in the USA using DNA profiling.

Uses of DNA Fingerprinting in Forensic Science:

•Because STRs and VNTRs are inherited from each of our parents, DNA profiling can also be used to establish paternity in cases involving custody and child support. Other uses in forensics include the identification of victims of catastrophes such as the September 11 attacks.

•It must be remembered that these tests are not always foolproof and should be used in conjunction with other evidence where possible. DNA fingerprinting has never the less affected the outcome of criminal investigations in a revolutionary way.


Centers for DNA Fingerprinting:

•Department of Biotechnology.•(M/o Science & Technology, Government of India)CDFD, Office Block :Bldg. 7, Gruhakalpa, 5-4-399 / B, Nampally, Hyderabad - 500 001 Laboratory Block :Tuljaiguda (Opp MJ Market), Nampally, HyderabadPh.: 040-27151344Fax: 040-27155610Website: www.cdfd.org.in •The diagnostic screening at CDFD is operated from the Nizam's Institute of Medical Sciences (NIMS)Punjagutta, Hyderabad, Andhra Pradesh, India. Phone Number : +91-40-23489000 Fax Number: +91-40-23310076

Conclusion:

With a beginning as a mere forensic tool, the world of DNA fingerprinting has a gone a long way in revealing the genetic identity of living beings. With massive evolution in concepts and techniques it has given a lot to various fields cutting across the spectrum. In plants it has not only helped in identifying species but also in defining a new realm in plant genomics, plant breeding and in conserving the biodiversity. With world paving way for developments in biotechnology, DNA fingerprinting promises a very powerful tool in our future endeavors.

DNA Fingerprinting

Education

dna dna strands

length dna

lineage dna fingerprinting

dna evidence

dna database

dna samples

cutting of dna

individuals dna