molecular identification technique

1. Molecular identification technique
(Blotting techniques)
Dr. Maryam Sabah naser
advance laboratory technique (8)

2. Blotting techniques :-Blotting is the technique in
which nucleic acids or proteins are immobilized onto
a solid support generally nylon or nitrocellulose
membranes. Blotting of nucleic acid is the central
technique for hybridization studies. Nucleic acid
labeling and hybridization on membranes have
formed the basis for a range of experimental
techniques involving understanding of gene
expression, organization, etc.

3. Identifying and measuring specific proteins in complex
biological mixtures, such as blood, have long been
important goals in scientific and diagnostic practice.
More recently the identification of abnormal genes in
genomic DNA has become increasingly important in
clinical research and genetic counseling. Blotting
techniques are used to identify unique proteins and
nucleic acid sequences. They have been developed
to be highly specific and sensitive and have become
important tools in both molecular biology and clinical
research.

4. General Procedure for blotting
Homogenization of the sample which involves the
purification of DNA/RNA/proteins which is performed after
extraction from a variety of sources such as cells or tissue.
Digest the DNA with restriction enzymes into fragments,
which is not required for RNA(northern blot).
Separation of the molecule of interest by an electrophoresis
membrane generally on an agarose gel for DNA fragments.
In the case of RNA samples they can be separated on an
agarose gel in presence of formaldehyde as the denaturing
agent. This is necessary as formaldehyde confines secondary
structures of RNA molecules.
Transferring the molecules (DNA/RNA fragments) to a nitro
cellulosic membrane/ nylon membrane from the gel.

5. Prehybridization (Blocking): Washing of the nylon membrane with
a prehybridization or blocking solution comprising salmon sperm DNA is
required inorder to block non-specific DNA interactions and also this
helps in the reduction of background noise. As an alternative, there
are some commercially available blocking buffers loke PerfectHyb™
Plus buffer in which there is no requirement of salmon sperm DNA for
blocking purpose.
Note:-For the preparation of probe fresh probe DNA labeled
with 32P alpha-labeled dCTP is prepared.
Hybridization or identification of the molecule which is achieved
by incubating the blot with the specific labeled probe.
For the detection of the probe and the sequence of interest DNA/RNA
the film is exposed to -80°C.

6. After being transferred to the support medium the
immobilized protein or nucleic acid fragment is localized
by the use of probes, such as antibodies or DNA, that
specifically bind to the molecule of interest. Finally, the
position of the probe that is bound to the immobilized
target molecule is visualized usually by autoradiography.
Three main blotting techniques have been developed and
are commonly called Southern, northern and western
blotting.

7. Southern blot :-is a method used to check for the presence of
a DNA sequence in a DNA sample.
A restriction enzyme is used to cut a sample of DNA into fragments that
are separated using gel electrophoresis. The DNA fragments are
transferred out of the gel to the surface of a membrane. The membrane
is exposed to a DNA probe labeled with a radioactive or chemical tag.
If the probe binds to the membrane, then the probe sequence is
present in the sample.

9. Applications
1. Southern blots are used in gene discovery, mapping ,
evolution & development studies , diagnostics &
forensics .
2. Deletions / insertions .
3. pointmutations / polymorphisms .
4. Structural rearrangements .
5. Allow for determination of molecular weights of
restriction fragments .
6. Presence of particular bit of DNA in the sample.

10. Northern blot:-technique is used to study gene expression
by detection of RNA (or isolated mRNA) in a sample. With
northern blotting it is possible to observe cellular control over
structure and function by determining the particular gene
expression levels during differentiation, morphogenesis, as
well as abnormal or diseased conditions. This technique was
developed in 1977 by James Alwine, David Kemp and
George Stark at Stanford University.

11. General Procedure for blotting
1.Homogenize the sample.
2.Separation of the molecule of interest by an electrophoresis
membrane.
3.Transferring the molecules to a nitro cellulosic membrane/ nylon
membrane.
4.Hybridization or identification of the molecule
Northern blotting takes its name from its similarity to the first
blotting technique, the Southern blot. The major difference is
that RNA, rather than DNA, is analyzed in the northern blot.

13. Applications
1. A standard for direct study of the gene expression at the
level of mRNA .
2. Detection of mRNA transcript size .
3. Study of RNA splicing – can detect alternatively spliced
transcripts .
4. Study RNA half life

14. western blot (alternatively, immunoblot) :- is used to detect
specific proteins in a given sample of tissue homogenate or
extract. The method originated from the laboratory of George
Stark at Stanford. The name western blot was given to the
technique by W. Neal Burnette.

17. Applications
1. The confirmatory HIV test employs a western blot to detect
anti HIV antibody in a human sample .
2. Proteins from known HIV infected cells are separated &
blotted on a membrane then the serum to be tested
3. is applied in the primary antibody incubation step. Free
antibody is washed away & a second anti human
antibody linked to an enzyme signal can be added

18. What’s the difference between a Southern, Northern, and Western Blot?

19. Microarray is a technique which holds promise and has an exceptional
sensitivity and the capacity to detect several pathogens simultaneously.
DNA microarray ( DNA chip or biochip) is a collection of microscopic DNA
spots attached to a solid surface. Scientists use DNA microarrays to
measure the expression levels of large numbers of genes simultaneously
or to genotype multiple regions of a genome.
However, microarrays are currently too expensive to be adapted for routine
diagnostics, and their diagnostic use requires broad-based nucleic acid
amplification prior to analysis which is not well established.

20. Note :-Laboratory results should always be interpreted in the context of the clinical
presentation of the patient, and appropriate site, quality, and timing of specimen
collection are required for reliable test results.
Several molecular methods can be used for genotyping, which allows the
identification of different subtypes of the pathogen; genotyping plays a role
in the risk assessment and management of infections.
Clinicians need to recognize the enhanced accuracy and speed of the
molecular diagnostic techniques for the diagnosis of infections, but also to
understand their limitations.

21. There are 2 types of DNA Chips/Microarrays:
1.cDNA based microarray
2.Oligonucleotide based microarray
There are certain requirements for designing a DNA
microarray system:
1.DNA Chip
2.Target sample (Fluorescently labelled)
3.Fluorescent dyes
4.Probes
5.Scanner

22. The principle of DNA microarrays lies on the hybridization between
the nucleic acid strands.
•The property of complementary nucleic acid sequences is to specifically
pair with each other by forming hydrogen bonds between complementary
nucleotide base pairs.
•For this, samples are labeled using fluorescent dyes.
•At least two samples are hybridized to chip.
•Complementary nucleic acid sequences between the sample and the probe
attached on the chip get paired via hydrogen bonds.
•The non-specific bonding sequences while remain unattached and washed
out during the washing step of the process.

23. • Fluorescently labeled target sequences that bind to a probe sequence
generate a signal.
• The signal depends on the hybridization conditions (ex: temperature),
washing after hybridization etc while the total strength of the signal,
depends upon the amount of target sample present.
• Using this technology the presence of one genomic or cDNA sequence in
1,00,000 or more sequences can be screened in a single hybridization.