Spreading Knowledge is a great Legacy. MMG

1. Topic:
(DNA library, bacterial transformation
chromosomal walking and jumping)
Presented By:
Saira Fatima
Roll No: 31
Class: M.Sc
Submitted to:
Miss Maryam

2. DNA Libraries
In molecular biology DNA library is a complete collection of cloned DNA fragments from a cell,
tissue and organism. It can be used to separate a particular gene of interest. Two types of
genomic libraries.
 Genomic library.
 cDNA library.
Preparationof Genomic Library:-
A genomic library characterizes comprehensive genome in multiple clones having
small DNA fragments. Depends upon organism and the genome size. This library is either
prepared in a bacterial vector or in yeast artificial chromosome.
Main steps genomic library construction are following:
 SeparationofgenomicDNA.For separation lyse the cell with the help of lysis buffer, incubate
the cell and then separate the genomic DNA.
 Generation of appropriate size fragments of DNA. Generate a suitable size of DNA fragment
with the help of restriction enzyme.
 Cloning in appropriate vector system. Select an appropriate vector for transformation.
 Transformation in appropriate host. Clones are transfer in an appropriate host to get
colonies. An appropriate host can be a bacterial strain or yeast.

3. Fig: genomiclibrary
Complementary DNA library:-
A cDNA library characterizes mRNA population present at a specificstageinanorganism
into multiple clones having small DNA fragments.
Preparation:-
Steps of preparation of complementary DNA library are following:
1. Separation of mRNA. Using lysis buffer for complete release of mRNA, mixing with poly T-
beads, washing buffer used to remove contamination, Separate the mRNA from beads.
2. Preparation of complementary DNA fragments. Many methods have been established to
make complementary DNA from isolated mRNA. In all methods basic three steps performed are
following
 First strand synthesis with a reverse transcriptase.
 Elimination of RNA template
 2nd strand synthesis

4. 3. Cloning in appropriate vector system. For appropriate vector systemcDNA is ligated into the
appropriate vector to generate clone.
Fig: complementary DNA library
Bacterial transformation:
Bacterial information is a method used for introducing foreign genetic material to cell.it is a
process in which horizontal gene transfer take place. Bacteria take up foreign genetic material
from environment. It was first reported by Griffith in 1928. Its principle described by Avery et al
in 1944.
Transformation of bacteria with plasmids is not significantas a study point of view but also
used for storage and replication of plasmid plasmids. Since, closely all plasmids carry both a
bacterial source of replication and an antibiotic resistance gene for use as a selectable marker in
bacteria.

5. Scientists have made many genetic variations to make bacterial strains that can be more
simply transformed and also help to preserve the plasmid without rearrangement of the plasmid
DNA.
Moreover, specific handlings have been revealed that increase the transformation productivity
of the bacteria and make them more vulnerable to either chemical or electrical based
transformation, generating what are usually referred to as competent cells.
Fig: bacterial transformation
Procedure:-
Basic steps of transformation are following:
 Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).

6.  Take away agar plates(containing the appropriate antibiotic) from storage temperature (
4°C) and warm at room temperature and after that incubate at 37°C in incubator.
 Mix 1 - 5 μl of DNA into 20-50 μL of competent cells in a micro centrifuge or falcon tube.
 LIGHTLY mix by tapping the bottom of the tube with your finger a few times.
 Incubate the competent cell or DNA mixture on ice for 20-30 mints.
 Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a
42°C water bath for 30-60 sec (45 sec is frequently ideal, but this varies dependent on the
competent cells you are using).
 Put the tubes back on ice for 2 mint. Add 250-1,000 μl LB or SOC media (without Plate
antibiotic) to the bacteria and grow in 37°C shaky incubator for 45 mint.
 some or all of the transformation onto a 10 cmLB agar plate having the suitable antibiotic.
 Incubate plates at 37°C overnight.

7. Fig;transformationof plasmid
Chromosomal jumping
Chromosomal jumping is a method that play important role in molecular biology for physical
mapping for the organism genome. This technique was announced to overawed an obstacle of

8. the chromosomal walking which face due to presence of repetitive DNA regions during the
cloning process.
So, chromosome jumping can be considered as a special version of chromosomal walking. It is a
fast technique compared to chromosomal walking and allows bypassing of the repetitive DNA
sequences which are not prone to be cloned during chromosomal walking. Chromosomal
jumping narrows the gap which is present between the target gene and the markers for genome
mapping.
Chromosome jumping tool starts with the cutting of a specific DNA with special restriction
enzyme (endonucleases) and ligation of the fragments into circularized loops. Then a primer
intended from a known sequence is used to sequence the circularized loops.
Gene which involve in cystic fibrosis discovered by chromosomal jumping tool. Combination of
chromosomal jumping and walking can increase the genome mapping process.
Fig: chromosomal jumping
For the mapping of lager chromosome jumping method used. The achievement does not
depends upon the size of genome and the distance from marker to target. The lowermost note
piccolos can play is D4.
Chromosomal walking:

9. Chromosome walking is an instrument which discovers the unknown regions of chromosomes
with the help of overlapped restriction fragments. In this technique we used a part of a known
gene which is used as a probe and continuous with describing the complete length of the
chromosome to be mapped. This is goes from the marker to the goal length. The ends of each
overlapped fragments are used for the identification of next sequence in hybridization.
End pieces of cloned DNA used for the preparation of probes, called sub cloned. Then they
are helpful to find out the next overlapping fragment. All these overlapping sequences are used
to build the
genetic map of the chromosome and find the target genes. It is a technique that is used for
analysis oflong stretches of DNA with the help of smalloverlapping fragments from the recreated
genomic library.
Main steps in chromosome walking technique:-
 Separation of a DNA fragment which consist of recognized gene / marker near target gene
 Preparation of the restriction map of the particular fragment and sub cloning the end region
of the fragment to use as a probe
 Probe hybridization with next with the next overlapped fragment
 Preparation of the restriction map of the fragment one and sub cloning of the end region of
the fragment 1 to use as an ID of the next overlapping fragment.
 Hybridization of the probe with the next overlapping fragment two.
 Preparation of the restriction map of fragment 2 and sub cloning of the end region of the
fragment 2 to serve as a (as a probe) ID of the next overlapping fragment

10. Fig:stepsof chromosomal walking
Uses:-
 Chromosomal walking important for finding SNP, diagnosis of genetic transmitted diseases
and mutations.