The methods used for DNA finger printing are the same Molecular markers...so for detailed note on the steps which is explained in DNA typing can be used to study the performance pf markers too...

1. AFSATH.B
M PHARM 1ST YEAR
PHARMACOGNOSYAND
PHYTOCHEMISTRY

2. Developed by Dr. Alec Jaffrey in 1984
A small set of DNA variation that is very likely to be different in all unrelated individual, thereby being as
unique to individuals as are fingerprint.
o “DNA fingerprinting is a quick way to compare the DNA sequence of any two individual”
o Used especially for identification by extracting and identifying the base pair pattern of an individual’s
DNA
o DNA typing, DNA profiling, DNA testing and Genetic finger printing

3. STEPS
1. Sample collection
2. Isolation of DNA
3. Restriction digestion
4. Separation of DNA sequence
5. Southern blotting
6. Hybridization
7. Autoradiography

5. II. ISOLATION OF DNA
1. 1gm of leaf is taken and powdered using liquid nitrogen.
2. Transfer to a centrifuge tube containing extraction buffer(Cetyl Trimethyl Ammonium Bromide (CTAB) ,
NaCl, EDTA,TrisHCl , β mercapto ethanol)
3. Centrifuge
4. Treated with chloroform : Isoamyl alcohol(24:1) & centrifuge
5. Supernatant is taken and treated with cold isopropanol to precipitate DNA.
6. The precipitate further treated with 70%alcohol to further precipitate the DNA
7. Stored at 40 c
III. AMPLIFICATION
• Done by PCR
• Steps involved in PCR:-
1. Denaturation
2. Annealing
3. Extension/ Elongation

6. 1. DENATURATION
When a DNA solution is heated enough,
The double-stranded DNA unwinds, and the
Hydrogen bonds that hold the two strands together
weaken and finally break. The process of breaking
a double-stranded DNA into single strands is
known as DNA denaturation, or DNA melting.
2. ANNEALING
• Temperature of reaction mixture is cooled to
45-60˚C
• Primers base pairs with complementary
sequence in the DNA
• Hydrogen bond reforms
• Annealing is fancy word of renaturing

7. 3. EXTENSION
• Temperature shifted to 72˚C- ideal for
polymerase
• Primers are extended by joining the base
complementary to DNA strands
• Elongation continues by the polymerase
which add dNTPs from 5’-3’ side
• Deoxynucleosides triphosphates (dNTPs)
required for the synthesis of DNA are
present in large excess
• synthesis step can be repeated a lot of
times.
• To withstand the repeated exposure to high
temperatures, a thermostable DNA
polymerase is used for PCR - usually Taq
polymerase.
• Taq polymerase works best at around 75
degrees centigrade.
• The time required for this stage depends
on the length of the target sequence (for
eg; the rate of primer elongation by Taq
polymerase is about 50 - 100
nucleotides/sec).

8. PCR CYCLE• The optimum number of cycles
depend on the initial
concentration of targeted
DNA.
• The increase in number of
cycles will increase the
complexity of non-specific
background product

9. IV. RESTRICTION ENDONUCLEASE DIGESTION
• Also called as molecular scissors
• DNA is cut into fragments with restriction enzyme(RE) based-
on nucleotide sequence
• Nowadays many artificial RE are also used
How does it work????
• Restriction enzymes attach to DNA and are activated by restriction sequences in the
DNA. Once activated, the restriction enzymes hydrolyse and destroy the bonds between
nucleotides. The restriction sequences along the DNA are inherited, thus, people who are
related have similar restriction sequences along their DNA.

10. V. SEPARATION OF DNA SEQUENCE
• Fragments are separated according to molecular size using agarose gel electrophoresis
• DNA fragments are injected into wells and an electric current is applied along the gel.
• Electrical current carries negatively-charged DNA through gel towards positive (red)
electrode
• Distance moved in given time will depend on mass of molecule of fragment
• The separated fragments are visualized by staining them(Ethylene bromide, methylene
blue, nile blue A) or by using complementary gene probe
{Gene probe:- single stranded piece of DNA with abase sequence complementary to the
DNA that you wish to identify and it must be labelled}

12. VI. SOUTHERN BLOTTING
• Separated DNA sequences are transferred on a nitro
cellulose or nylon membrane
• They stay immobile on the sheet. The sheet is then
incubated.

13. VII.HYBRIDIZATION
• The nylon membrane is immersed in a bath
• Radioactive probes are added
• These probes target a specific nucleotide sequence of the single DNA strand on the membrane that is
complimentary to them
• When the sequences of the target DNA probe are determined then it is labelled so that it can be identified.
The tagging is done by a fluorescent material so that it can be distinguished.

14. VIII.AUTORADIOGRAPHY
• When the probe is labelled, now it can be taken out of the nylon membrane and can be viewed through
autoradiography on the x-ray film.
• The nylon membrane is pressed on the x ray film
• Dark bands are developed at the probe site which resembles the bar codes

15. DIFFERENT METHODS OF DNA FINGERPRINTING
1.Restriction fragment length polymorphism (RFLP)
2. Randomly amplified polymorphic DNA (RAPD)
3. Amplified fragment length polymorphism (AFLP)
4.Simple sequence repeats (SSR)

16. APPLICATION….
o Forensic science
o Paternity and Maternity determination
o Personal identification
o Application to both plants and animals
o To identify genetic diversity with in breeding populations
o To differentiate between plant species cultivars
o To identify plants containing gene of interest
o To detect genetically modified organism in agriculture
o used for identify species or population
o used for estimating genetic distance and finger printing of wheat
o biological parentage
o used for characterization & determination of genetic diversity of tea germplasm

17. o Confirms the morphology and growth behavior of suspected hybrid .
o Effect of habitat fragmentation
o Extend of gene flow within and among populations.
o Reveal the extend of clonal growth
o Spontaneously occurring somatic mutation