Microarrays (DNA chips) allow large numbers of DNA probes to be immobilized on a solid surface like nylon or glass. They have various applications including identifying genes responsible for development, diseases, and single nucleotide polymorphisms. Restriction fragment length polymorphism (RFLP) detects length variations in DNA fragments after restriction enzyme digestion, revealing polymorphisms. DNA fingerprinting analyzes variable numbers of tandem repeats in satellite DNA, producing unique patterns that can identify individuals in forensic investigations.

1. Microarrays (DNA Chips)
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. Microarrays (DNA Chips)
• These are recent advancements for DNA sequencing
as result of advances made in automation
miniarization.
• A large no. of DNA probes, each one with difference
sequence, are immobilized at defined positions on the
solid surface, made up of either nylon or glass.

4. Applications of DNA chips
 There have been many successes with this relatively new
technology of DNA chips. Some of them are:-
I. Identification of genes responsible for the development of
nervous system.
II. Detection of genes responsible for inflammatory diseases.
III. Construction of microarrays for gene in the genome of E.coli
and almost all the genes of the yeast saccharomyces
cerevisiae.

5. Contd…
IV. Detection and screening of single nucleotide polymorphisms.
V. Rapid detection of microorganisms for environmental
monitoring.
VI. Expression of several genes in prokaryotes has been
identified.

6. Applications
1. Functional genomics In the functional genomics
area, DNA chips are used for measurement of
expression level of genes or expression patterns of
genes. A limited number of genes involved in a
pathway may be selected.
2. Diagnostics and genetic mapping DNA chips are
used for diagnositcs. For example, diagnostic chips
are prepared to detect mutant alleles in cystic fibrosis
and beta globin genes. DNA chips are also used for
genotyping of hepatitis C virus in blood samples.

7. 3. DNA sequencing by hybridization (SBH) DNA
sequence is determined by using DNA chips. These
DNA chips contain set of oligonucleotides of
particular size. Hybridization is carried out with DNA
of unknown sequence. Then hybridization pattern is
used to obtain DNA sequence.
4. Single nucleotide polymorphism (SNP) and point
mutations are detected using DNA chips. DNA chips
are used in Human Genome Project for detection of
point mutations.

8. 5. Proteomics DNA chips are useful in this area also.
DNA chips are used to identify genes involved in
protein-protein interactions.
6. Reverse genetics DNA chips are used in this area
where organism complete genome sequence is
known. It involves introduction of
deletions/insertions or substitutions at will
followed by analysis of their fitness.
7. DNA chips are used in genomic miss match
scanning (GMS).
8. Drug discovery and agriculture biotechnology are
other areas where DNA chips are currently in use.

9. Future of DNA chips
 Major limitation of DNA chips at present is the unavailability
of complete genome arrays for higher eukaryotes, including
humans.
 It is expected that within the next few years such DNA chips
will be available.
 This will help the biotechnologist to capture the functional
snapshot of the genome in action for higher organisms.

11. Restriction fragment length
polymorphism (RFLP)

12. Restriction fragment length polymorphism (RFLP)
• It is another technique based on hybridization principle.
• DNA is a polymorphic molecule, i.e.exist in several forms.
• DNA of an individual varies from others. Sequence of DNA
of an individual is unique.
• Further, mutations in DNA generates polymorphic DNA in
same individual, which occurs in diseases.
• So, DNA polymorphism is due to variations in sequence.
• When DNA of an individual is subjected to digestion with
restriction enzyme fragments of varying sizes or lengths
that are unique to individuals sequence or cell are
produced.

13. • RFLP may also result from presence of variable
numbers of tandem repeats (VNTR) in DNA.
• These are short sequences of DNA that are scattered
locations in genome and repeated in tandem.
• The number of these repeats are unique to individual.
• When DNA of two individuals is subjected to digestion
with restriction enzymes fragments that vary in length
and number are generated.
• Therefore, RFLP of two individuals results from the
differences in the location and number of cleavage sites.
• Differences in DNA of two individual may be due to
evolutionary changes.

14. • RFLP is similar to southern blotting in many aspects.
• Initial step of RFLP involves digestion of more DNA
samples with restriction enzymes where as in
southern blotting only one DNA sample is digested.
• Rest of the steps of RFLP are those of southern
blotting.
• Hence, in RFLP next step probes are used for
hybridization.
• Probes hybridizes with fragment containing
complementary sequences.
• Then polymorphisms are detected by presence or
absence of bands after hybridization.

16. Methods for detection of RFLPs
Two common methods are:
1. Southern hybridization
2. Polymerase chain reaction (PCR)

19. Applications
1. RFLP is used as a diagnostic test of inherited
disease. For example, HbS: In HbS gene there is
loss of one restriction site for restriction enzyme due
to mutation where as normal HbA gene has two
cleavage sites.
So, RFLP of sickle-cell anemia patient
shows two bands where as in RFLP of normal
individual three bands appear.

20. 2. RFLP is also used to identify chromosomal
difference.
3. RFLP is used for isolation and sequencing of closely
related genes.
4. RFLP is combination with PCR is used to detect
DNA variations.

22. DNA finger printing

23. DNA finger printing
It is the present day genetic detective in the
practice of modern medical forensics.
History:- original DNA fingerprinting technique was
developed by Alec Jaffreys in 1985. It is commonly
used a more general term DNA profiling is preferred.
 DNA fingerprint is an analysis of the nitrogenous base
sequence in the DNA of an individual.

24. DNA finger printing
• Use of finger prints in crime detection (forensic
science) is well known.
• Like finger prints, DNA of an individual is
characteristic of that individual.
• In other words, DNA make up of every human is
different.
• DNA finger printing is a technique used to identify an
individual from DNA obtained from blood or semen
or piece of tissue.

25. • The technique is based on identification of tandem
repeats of satellite DNA.
• Satellite DNA consist of repetitive sequence. For
example, satellite DNA of fruit fly consist of
repetitive sequence ACAAA CT.
• The length of repetitive sequence or satellite DNA
varies and depends on the organism or individual.
• In addition, satellite DNA is repeated several times
(107) and present on all chromosomes of genome

26. steps of DNA finger printing
1. DNA of suspect is cleaved with restriction
endonuclease, which does not cut satellite DNA.
2. The fragments are separated on agarose gel
electrophoresis and southern blotted.
3. Satellite DNA is identified by radiolabelled satellite
DNA probe.

27. 4. Probe hybridizes with DNA fragments containing
satellite DNA.
5. The number of satellite DNA present in a given
fragment depends on the size of fragment. The size of
fragment in turn depends on the individuals DNA. It
varies from individual to individual.
6. Southern-blot pattern thus obtained is unique to
individuals just like finger print and it is called as
DNA finger print. In Figure DNA finger prints of four
unrelated people are given. Note that each one has
characteristic pattern. With the help of this DNA
finger print, sample is matched to individual or
suspect.

29. Applications of DNA fingerprinting
 The amount of DNA required for DNA fingerprint is
remarkable small.
 The minute quantities of DNA from blood strains,
body fluids, hair fibre or skin fragments are enough.
 PCR is used to amplify this DNA for use in
fingerprinting.
 It has wide range of applications- most of them
related to medical forensics.

30. Some important are:
1. Identification of criminals, rapists, thieves etc
2. Settlement of paternity disputes.
3. Use in immigration test cases and disputes.
NOTE: DNA finger printing and parental dispute
• Since half of the child DNA comes from parental DNA,
DNA finger prints are used to establish parentage of child
by matching childs DNA finger prints with parental DNA
finger prints