describes various techniques for confirmation of gene transfer

1. DEEPALI RAHI
(2015-11-102)
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2.  Southern blotting
 Colony hybridization
 Dot blot technique
 Northern blotting
 Western blotting
 Polymerase chain reaction
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3.  A Southern blot - routinely used in molecular
biology to check for the presence of a DNA
sequence in a DNA sample
 Genomic DNA extracted from putative
transgenic individuals – transformation
 Based on – DNA-DNA hybridization
 Invented by – E.M. Southern
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4. 1.Extraction of genomic DNA -transgene
2.Restriction digestion
3.Electrophoresis of genomic DNA- AGE
4.Before blotting, treat the gel with
0.2N HCl or alkali - denaturation and
neutralization –single stranded DNA
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5. 5.Blotting- Transfer gel to the nitrocellulose
membrane
6. After the transfer, cross-link the DNA to the
nitrocellulose membrane
7. Making the probe-
Label with the probe to hybridize DNA using radioactive
/ non-radioactive methods
8. Exposed to x-ray film – visualize gene of interest
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7.  Technique to identify bacterial colony with
foreign gene – genetic engineering
 Transformed colonies are detected –
radioactive DNA/RNA used in the probe
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8.  Transformed colonies – agar plate – master
plate
 Colony of master plate are replica plated on
nitrocellulose membrane
 A reference point is marked on both for
further comparison
 After colonies appear - Alkali - lyse the
bacterial cell – denature DNA
 Filter – Proteinase K – digest & remove
protein
 Filter is baked at 80 ̊̊c – to fix the DNA –
impregnation
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9.  Hybridization with radioactive probe – DNA
sequence used in transformation
 Washing off – unhybridized probe
 Hybridized colonies – visualized by
autoradiography
 Only transformed colonies will show
autoradiograph
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11.  The mRNA produced by transgene is the most
readily detected if they have unique
sequences
 A high purity mRNA preparation is obtained
from appropriate tissue of transgenic
individuals
 Thus detection of RNA is technique to
confirm gene transfer – Northern blotting
hybridization or Dot-Blot technique
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12.  The northern blot is a technique used to
detect RNA
 RNA – separated by gel electrophoresis
 Bands are transferred to
diazobenzyloxymethyl (DBM) paper/ nylon
paper
 Probe hybridization – Single stranded DNA
complementary to RNA
 Autoradiography detection
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14. SOUTHERN BLOTTING NORTHERN BLOTTING
1. DNA is separated by gel
electrophoresis
1. RNA is separated by gel
electrophoresis
2. DNA has to be denatured before
AGE
2. This step is skipped
3. Nitrocellulose membrane is
generally used
3. DBM paper or nylon membrane is
generally used
4. Hybridization with probe is DNA-
DNA hybrid
4. Hybridization with probe is RNA-
DNA hybrid molecule
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15.  A dot blot is a technique - detect biomolecules
such as nucleic acid i.e DNA/RNA
 In this technique biolomolecules to be detected
are not first separated by electrophoresis – non
fractionated rather single test run
 Instead a mixture containing the molecule to be
detected is applied directly on the membranes
as a dot
 This is then followed by nucleotide probes
 Washing- to remove unhybridize probe
 Autoradiography – X-ray film
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17.  By detecting protein or gene expression we
can confirm its gene presence
 Proteins can be detected by their interaction
of antibodies, lectins or other compounds by
polyacrilamide gel
 Technique to detect protein – Western
blotting
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18.  The western blot or immunoblot - analytical
technique used to detect specific proteins in
a given sample.
 The proteins are electrophoresed in
polyacrilamide gel (PAGE) - transferred onto
a nitrocellulose membrane, by capillary
blotting or electrophoresis blotting (faster)
 Antibodies as probe (specific to the target
protein) or lectins or some other compounds
 Antibodies or probes are radioactive
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19.  Species specific secondary antibody is added
– sandwich reaction
 Secondary Ab binds to primary Ab which is
bind to protein band
 Secondary antibody is labeled with enzyme,
radioactive or fluorescent tags
 A single preparation of labeled molecule can
be employed as detection of various probes
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21.  Given by – Kary mullis – 1983
 The polymerase chain reaction (PCR) -
technique used in molecular biology to
amplify a single copy or a few copies of a
piece of DNA across several orders of
magnitude- generating thousands to millions
of copies of a particular DNA sequence
 Use to confirm transformed DNA by using
specific primer to suspected DNA
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22.  Initially make master mix – mix DNA segment
to be amplified, two primer molecule, dNTP,
DNA polymerase enzyme, Mg++ in
appropriate quantity
 DENATURATION – 90-94 ̊c- 2 minutes – single
stranded
 ANNEALING – primer is annealed to
complementary DNA – 40-60 ̊c for one minute
 PRIMER EXTENSION – DNA polymerase enzyme
synthesizes complementary strand using 3΄OH of
the primers
 Cycle is repeated
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