Searching the genes of interest in a DNA library

1. M.SRIDEVI
II.MSC (MICROBIOLOGY)
18PY15
LIBRARY SCREENING

2.  DNA library is a collection of DNA
fragments. It may be divided into two types.
 The genomic library contains DNA
fragments representing the entire genome of
an organism.
 The cDNA library contains only
complementary DNA molecules synthesized
from mRNA molecules in a cell.
Introduction

3.  The genomic library is normally made by λ phage vectors, instead of plasmid vectors,
for the following reasons.
 The entire human genome is about 3 x 109 bp long whereas a plasmid or λ phage
vector may carry up to 20 kb fragment. This would require 1.5 x 105 recombinant λ
phages.
 The maximum number to allow isolation of individual colonies is about 200 colonies
per dish. Thus, at least 700 petri dishes are required to construct a human genomic
library.
 By contrast, as many as 5 x 104 λ phage plagues can be screened on a typical petri
dish. This requires only 30 petri dishes to construct a human genomic library.
 Another advantage of the λ phage vector is that its transformation efficiency is about
1000 times higher than the plasmid vector.
Genomic Library

4. f
Preparation of a DNA Library
Fig: Preparation of the genomic library using λ
vectors. It is basically the cloning of all DNA
fragments representing the entire genome

5. cDNA Library
 The advantage of cDNA library is that it contains only
the coding region of a genome.
 To prepare a cDNA library, the first step is to isolate
the total mRNA from the cell type of interest. Because
eukaryotic mRNAs consist of a poly-A tail, they can
easily be separated.
 Then the enzyme reverse transcriptase is used to
synthesize a DNA strand complementary to each
mRNA molecule.
 After the single-stranded DNA molecules are converted
into double-stranded DNA molecules by DNA
polymerase, they are inserted into vectors and cloned.

6. Screening procedures
Screening
Colony and plaque hybridization
Expression screening
Hybrid arrest and release

7. Screening
The process of identifying one particular clone containing the gene of interest
from among the very large number of others in the gene library .
The identification of specific clone from a DNA library can be carried out by
using either
(1)the sequence of the clone or
(2)the structure/function of its expressed product
Screening the product of a clone is applied only to expression libraries where the
DNA fragment is expressed to yield proteins and the product is recognized by
antibody /ligand

8. Screening libraries
• Searching the genes of interest in a DNA library
• Hybridization to identify the interested DNA or its RNA product
1. Radiolabeled probes which is complementary to a region of the
interested gene
2. Hybridize the labeled probe with DNA membrane (Southern) or RNA
(Northern) membrane.

9. Hybrid arrest and screen
• Individual cDNA clones or pools of clones can be used to hybridize to mRNA
preparation
Hybrid arrest :
• Translate the mRNA population directly, and the inhibition of translation of some
products detected.
Hybrid release translation :
• Purify the hybrids and the hybridized mRNAs released from them and translated,
it identifies the protein encoded by the cDNA clone

10. I. Isolating individual clones
II. Screening by sequence
• Hybridization
• PCR
III. Screening by protein
structure/biological function
Screening libraries for specific genes

11. Screening by hybridization
 Very fast
 Applicable to a large number of clones
 Can identify clones that are not full length

12. Fig :Colony hybridization

13. Fig: Plaque hybridization

14. SCREENING BY PCR
The PCR is widely used to isolate specific DNA sequences from genomic DNA
and now It has been a useful technique for library screening.
This method is first demonstrated by takumi and lodish in 1994
To isolate a specific clone the PCR is carried out with gene specific primers that
flank a unique sequence in the target.
Pools of clones are maintained in multiwell plates.
Each well is screened by the PCR and positive wells are identified

15. EXPRESSION LIBRARIES SCREENING
 Identify the protein product of an interested gene
 If a DNA library is established using expression
vectors, each individual clone can be expressed to
yield a polypeptide.
 This type of screening is important where the DNA
sequence of the target sequence is unknown.
1. Protein activity
2.Western blotting using a specific antibody

16. Expression screening
• Antibodies can be used to screen the expression library.
Procedure :
‘Plaque lift’ ( taken by placing a membrane on the dish of plaque)
Immersed in a solution of the antibody
Detected by other antibodies
Repeat cycles of screening to isolate pure plaques

17.  Developed in 1970 when plasmid vectors are
used to construct genomic libraries
 Immunological screening involves the use of
antibodies that specifically recognize antigenic
determinants on the polypeptide
 This technique can be applied to any protein for
which an antibody is available.
 The molecular target for recognition is
generally an Epitope.
IMMUNOLOGICAL SCREENING

18. Earlier immunoscreening methods
employed radio-labeled primary antibodies
to detect antibody binding to the
nitrocellulose sheet
 It is now superseded by antibody
sandwiches resulting in highly amplified
signals.
The secondary antibody recognizes the
constant region of the primary antibody &
is, additionally, conjugated to an easily
assayable enzyme (e.g. horseradish
peroxidase or alkaline phosphatase) which
can be assayed using colorimetric change
or emission of light using X-ray film .

19. procedure
In this technique, the cells are grown as
colonies on master plates and transferred to a
solid matrix.
 These colonies are subjected to lysis releasing
the proteins which bind to the matrix.
 These proteins are treated with a primary
antibody which specifically binds to the protein
(acts as antigen). The unbound antibodies are
removed by washing.

20.  A secondary antibody is added which specifically binds to the
primary antibody removing the unbound antibodies by washing.
 The secondary antibody carries an enzyme label (e.g., horse radish
peroxidase or alkaline phosphatase) bound to it which converts
colorless substrate to colored product. The colonies with positive
results (i.e. colored spots) are identified and sub cultured from the
master plate)

21. Fig: Schematic process of immunological screening using antibody sandwich.