Screening of recombinants: blotting techniques, colony lift, immunological methods, south-western screening etc.

1. LADY AMRITBAI DAGA & Smt. RATNADEVI
PUROHIT COLLEGE SHANKAR NAGAR NAGPUR
(RAMABAI BARLINGAY SCHOOL OF
BIOTECHNOLOGY)
TOPIC:SCREENING OF RECOMBINANTS : Screening by complementation
,southern blotting, northern blotting, western blotting, colony lift,
immunoprecipitation, south-western screening.
PRESENTED BY: Vaishnavi R. Ghugal
M.Sc. Biotechnology( SEMESTER 2)
Batch 2021-22

2. CONTENTS
 INTRODUCTION
 SCREENING BY COMPLEMENTATION
 COLONY LIFT
 SOUTHERN BLOTTING
 NORTHERN BLOTTING
 WESTERN BLOTTING
 SOUTH-WESTERN SCREENING
 IMMUNOPRECIPITATION
 REFERENCE

3. INTRODUCTION
 SCREENING: A population of viable cells is subjected to some sort of analysis that
enables the desired sequence to be identified.
SCREENING METHODS
DIRECT
METHOD
Use of
marker or
reporter
gene
Selection by
complement
ation or
nonsense
suppression
Marker
inactivatio
n
techniques
INDIRECT
METHOD
Restriction
enzyme cleavage
pattern
Hybridization
techniques
Colony or
plague
hybridization
Colony
lift
Blotting
technique
s
Southern
blotting
Northern
blotting
Western
blotting
Detection of specific
proteins
Protein
synthesis in
mini cells
Immunologica
l method

4. SCREENING BY COMPLEMENTATION
 Functional complementation assay is an in vivo that is widely used to elucidate the
function/role of genes/enzymes.
 This technique is very common in biochemistry, genetics and many other disciplines.
 EXAMPLES:
 Blue-white screening(α complementation of β-galactosidase)

5. BLUE-WHITE SCREENING(α complementation
of β galactosidase)
Scientist discovered that deleting a section from lacZ gene
(a mutation called lacZΔM15) creates a nonfunctional
β-galactosidase enzyme. Providing DNA encoding this
section of amino acid(called α-peptide) to a lacZΔM15
mutant bacterial cell in trans complements the mutation
allowing for a functional enzyme. This process
is called α -complementation.
BLUE COLONY= NON-RECOMBINANT
WHITE COLONY=RECOMBINANT
Foreign DNA in MCS, no α fragment
No α fragment, no β -gal
No β -gal, no blue color(white colonies)
http://www.sigmaaldrich.com/content/dam/sigma-
aldrich/articles/biology/blue-white-screening/typical-blue-white-screening-
procedure.jpg

6. COLONY LIFT
 Colony hybridization is a screening method used in the selection of bacterial colonies
with a desired DNA sequence with high density.
 The procedure of colony hybridization was first developed by Grunstein and Hogness
in 1975. It is also called colony blot hybridization, colony lift or replica plating.
https://tse2.mm.bing.net/th?id=OIP.ZXDTVTS7cX8JIXY-eQkMAgHaFj&pid=Api&P=0&w=220&h=165

7. SOUTHERN BLOTTING
 Southern blotting technique is employed for the:
I. detection of specific DNA sequences/genes
II. detection of abnormalities in a given gene structure
 The method was named in honor of the British biologist Edwin M. Southern,
University of Oxford who first published it in 1975, and hence the name Southern
Blotting.
Major steps involved may be summarized as follows:
 Step 1: Extraction and Purification of DNA from cells.
 Step 2: Restriction Digestion (Fragmentation of sample).
 Step 3: Electrophoretic separation of DNA fragments in the sample.
 Step 4: Partial depurination to promote higher efficiency transfer of DNA fragments.
 Step 5: Denaturation with mild alkali and subsequent blotting.
 Step 6: Probe-hybridization (binding of analytical probe) to target molecule.
 Step 7: Visualization of the bound probe by Autoradiography

8. APPLICATIONS
 It is a widely accepted analytical technique used in
molecular biology and immunogenetics to identify the
DNA of interest from a mixture of DNA samples or to
detect a specific base sequence within a strand of DNA.
 Identification of methylated sites in particular genes.
 Preparation of RFLP maps.
 Detection of point-mutations, deletions or gene
rearrangements in DNA.
 In forensic medicine, for criminal identification and DNA
fingerprinting (VNTR).
 Detection and identification of transgene in transgenic
individuals.
 For diagnosis of infectious diseases and prognosis of
cancer.
 Useful in prenatal diagnosis of genetic diseases.
https://www.onlinebiologynotes.com/wp-content/uploads/2017/12/southern-blotting.gif

9. NORTHERN BLOTTING
 Northern blotting is used in molecular biology to study gene expression by detection of
RNA (isolated mRNA) in a sample.
 It is a variant of Southern Blotting, developed in 1977 by James Alwine, David Kemp,
and George Stark at Stanford University, with significant contributions from Gerhard
Heinrich.
Major steps involved Northern Blotting :
• Step 1: Extraction of total RNA from a homogenized tissue sample or from cells.
• Step 2: Electrophoretic separation of RNA fragments in the sample.
• Step 3: Denaturation to limit the secondary structure followed by subsequent blotting.
• Step 4: Probe-hybridization (binding of analytical probe) to target molecule.
• Step 5: Visualization of the bound probe by Autoradiography.

10. APPLICATIONS
 Used in diagnosis of environmental stress levels
and pathogen infection.
 To detect the over-expression of oncogenes and
down-regulation of tumor-suppressor genes in
cancerous cells when compared to 'normal' tissue.
 Used to identify gene expression in the rejection of
transplanted organs.
 Helpful in studying mechanism of RNA
degradation and splicing.
 Detection of molecular weight of a specific
mRNA.
 Identification of mRNA produced by transgenes to
protect the recombinants.
https://s-media-cache-
ak0.pinimg.com/originals/4d/df/37/4ddf37f5a902dd879bee3c1bda2a7bd5.jpg

11. WESTERN BLOTTING
 Western blotting is a widely applied analytical technique employed in molecular
biology and immunogenetics to identify and characterize specific proteins within a
sample of tissue homogenate or extract.
 The method by itself originated in 1979 in the laboratory of Harry Towbin (Friedrich
Miescher Institute, Switzerland), but the term ‘western’ was later put forth by W.
Neal Burnette in 1981.
Major steps involved may be summarized as follows:
 Step 1: Extraction and homogenization of the protein sample.
 Step 2: Treatment with a suitable buffering solution.
 Step 3: Electrophoretic separation of protein using SDS-PAGE.
 Step 4: Electro-transfer into nitrocellulose/PVDF filter membrane.
 Step 5: Preparation of membrane for antibody-staining.
 Step 6: Incubation with primary antibodies, specific to detection of target protein.
 Probe-hybridization (binding of analytical probe) to target molecule.
 Step 7: Detection via (i). Colorimetry, (ii). CCD Camera, LED/Infrared Imaging.

12. APPLICATIONS
 It is a routine method in molecular biology,
biochemistry, cell biology and
immunogenetics with a multitude of
applications.
 Widely used method for detection of target
protein from complex samples, using
antibody-based probes.
 Used to obtain information about quantity,
molecular weight and post-translational
modifications of proteins under study.
 It is an essential tool for analyzing protein
moieties of complex systems.
 Monitors changes in expression or post-
translational modifications in samples of
protein.
https://cdn.antibodies.com/image/resources/Western%20Blot%20-%20Figure%201.png

13. SOUTH-WESTERN SCREENING
 South-western screening is a method for identifying a protein which binds to a protein-
binding site on the DNA .
cDNA is cloned into suitable expression vector to form
expression library.
Library is plated on nutrient agar.
Well separated phage plagues are formed.
Nitrocellulose filter is applied on agar & then peeled of to
give imprint of the phage.
Foreign cDNA sequence are expressed to give protein, and the
fusion proteins are adsorbed in nitrocellulose membrane.
Membrane is incubated in radiolabelled dsDNA representing
the known protein -binding sequence.
DNA probe binds to specific protein. This leads to
identification of specific binding protein.

14. IMMUNOPRECIPITATION
 Immunoprecipitation is based on the use of antibodies to isolate the proteins against
which they are directed.
 The process is as follows:
Cells are incubated
with radioactive
amino acids to
label the proteins.
Radiolabelled cell extract is
incubated with the antibody
against the target protein.
The antibody
binds to specific
protein forming
antigen-antibody
complex.
The antigen-antibody
complex is isolated by
incubating with beads
that bind the antibody.
Beads are
boiled to
dissociate
complex.
Proteins
recovered
are
analyzed by
gel
electrophor
esis.
Radioactive
proteins can be
detected by
autoradiography.

15. REFERENCE
 C.B.POWAR page no.1556,1557
 Trayhurn, P. (1996) Northern Blotting. Pro. Nutrition Soc. 55:583-589.
 LINKS:
 https://youtu.be/bUIxNnfmH5k
 https://youtu.be/CMGbaItoE8M
 https://youtu.be/Igqc_mGmbVQ
 https://youtu.be/8qGYdGLbwpc
 https://youtu.be/MVuKxKvniME
 WEBSITES
https://microbenotes.com/southern-blot-principle-steps-and-
applications/#principle-of-southern-blot