The document discusses DNA fingerprinting techniques for identifying plants at the molecular level. It describes methods such as PCR-based techniques like RAPD, ISSR, and SSR, as well as non-PCR based RFLP. The key steps are DNA isolation from plant tissues, amplification using PCR, restriction enzyme digestion, gel electrophoresis, and comparing banding patterns to determine differences between individuals. DNA fingerprinting can be used for applications like criminal investigations by matching DNA from crime scenes to suspects.

2.  INTRODUCTION:
 The DNA fingerprinting unlike the usual
fingerprinting which is based on the
morphological features and primarily
restricted to humans is revealing the identity
of an organism at the molecular level.
 In fact this is the technique of finding the
genetic identity.
 This is primarily based on the polymorphisms
occurring at the molecular level, that is on
the base sequences of the genome.

3.  METHODOLOGY:
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.

4.  DNA ISOLATION:
High molecular weight DNA from plant
tissue can be isolated in a number of ways.
All methods involves basic steps of removal of all
cell wall and nuclear membrane around the DNA
and the separation of DNA from other cell
components such as cell debris, proteins, lipids
or RNA without affecting the integrity of the
DNA. The most commonly preferred method is
CTAB method.

5. PCR:
The DNA amplification by thermal cycling
called Polymerase Chain Reaction is in vitro
method that can be used to amplify a specific
DNA segment from small amounts of DNA
template or duplex into millions of copies. It was
invented by Kary Mullis.
Steps involved in PCR are:
 Heat Denaturation.
 Annealing.
 Primer Extension.

6. NON-PCR:RFLP
 To discover RFLPs, restriction enzymes (RE) are
used to cut DNA at specific 4-6 bp recognition
sites.
 Sample DNA is digested with one or more RE’s
and resulting fragments are separated according
to molecular size using gel electrophoresis.
 After that Southern Blotting is performed where
fragments are immobilized on a nylon
membrane, followed by hybridization to a labeled
probe that identifies the locus under
autoradiograph.
 Fragment sizes of digested DNA will differ
depending on the presence or absence of the
proper recognition sequence for the enzyme.

7. Southern blot
1. Restriction enzyme digestion
2. Agarose gel electrophoresis
3. Transfer DNA onto membrane
4. Hybridized with probe
RFLP
Restriction
Enzyme site 1. PCR amplify using
fluorescent primers
2. Capillary electrophoresis
RFLP-based
DNA fingerprinting
PCR-based
DNA fingerprinting
PCR product of different sizes
Fluorescent peaks of different mobilit
Radioactive bands
of different mobility

8.  CONTD….
 Differences in fragment length result from base
substitutions, additions, deletions or sequence
rearrangements within RE recognition sequences.
 Although two individuals of the same species have
almost identical genomes, they will always differ at a
few nucleotides.
 Some of these differences will produce new restriction
sites (or remove them), and the banding pattern seen
on a genomic Southern will thus be affected.
 For any given probe (or gene), it is often possible to
test different restriction enzymes until one which
gives a pattern difference between two individuals is
found, that is, a RFLP.
 The less related the individuals, the more divergent
their DNA sequences.

9.  RAPD:
 In RAPD analysis the total DNA from an organism
is mixed with (generally) 10 bp single stranded
DNA (primer) together with the four different
deoxynucleotides and a heat stable DNA
polymerase enzyme.
 The reaction mixture is placed on a thermocycler
(PCR-machine) which can change the
temperature of the reaction rapidly according to
a predefined PCR programme.
 Reannealing at low temperature makes the
primer molecules attach to complementary site
on the DNA.

10.  Elongation is the period when the polymerase
enzyme elongates the attached primers from
their 3' end. Denaturation separates the new
formed strands.
 After a few PCR cycles DNA pieces with well
defined length similar to the one between the
two original primer sites dominate the mixture.
This is because they amplify exponentially.
 In most RAPD reactions the result will be
amplification of 5 to 10 different DNA segments
whose lengths depend on the primer recognition
sites available in the genome.
 The DNA after amplification is visualized for
bands using Agarose Gel Electrophoresis (AGE).

11.  SSR:
 SSR analysis is amenable to automation
and multiplexing and allows genotyping to be
performed on large numbers of lines, and
multiple loci to be analyzed simultaneously.
 SSRs can be identified by searching among DNA
databases (e.g. EMBL and Gene bank), or
alternatively small insert (200-600bp) genomic
DNA libraries can be produced and enriched for
particular repeats.
 For separation and visualization of bands
Polyacrylamide Gel Electrophoresis (PAGE) is
conducted (6% acrylamide solution), stained and
the observed the bands.

12.  ISSR:
 ISSR analysis is technically simpler than any
other marker systems.
 The method provides highly reproducible results
and generates higher levels of polymorphism
compared to other markers in many systems.
 The advantage lies in long primer length.
 For separation and visualization of bands
Agarose gel electrophoresis is conducted.

13.  Use of RFLP in criminal investigation
Victim
SuspectA
SuspectB
SpermDNA
From crime scene
Matching of DNA
obtained from
Crime scene
Suspects

14.  ADVANTAGE:
 Criminal investigation
Matching of suspect DNA with that of crime scene
 Database of criminals’ DNA fingerprint
Matching of suspect DNA with criminal database
CODIS (Combined DNA Index system)
 Animal pedigree confirmation
Check authenticity of pedigrees of dogs, racing horses
 Diagnosis of inherited disorders
Any association between DNA fingerprints with genetic
diseases?
 Identification of dead bodies in natural disaster

15.  DISADVANTAGES:
 Labour intensive
 Expense
 Large quantity of DNA needed
 Highly sensitive to laboratory changes
 Cannot be used across populations nor across
species
 Can be technically challenging

16. DNA fingerprint