Transcript cDNA library

DEFINITION
WHAT IS GENOME?
• Genome is nothing but it contains all of the biological
information needed to build and maintain a living
example of that organism.
• The biological information contained in a genome is
encoded in its deoxyribonucleic acid (DNA) and is
divided into discrete units called genes.
WHAT IS GENOMIC LIBRARY?
• A genomic library is a population of host bacteria, each
of which carries a DNA molecule that was inserted into a
cloning vector, such that the collection of cloned DNA
molecules represents the entire genome of the source
organism.
Cloning Vectors
• A vector is used to amplify a single molecule of
DNA into many copes. A DNA fragment must be
inserted into a cloning vector. A cloning vector is
a DNA molecule that has an origin of replication
and is capable of replicating in a bacterial cell.
• Most vectors are genetically engineered plasmids
or phages. There are also cosmid vectors,
bacterial artificial chromosomes, and yeast
artificial chromosomes.
Plasmid Cloning Vectors
• Plasmids are circular, double-stranded
DNA molecules that exist in bacteria and in
the nuclei of some eukaryotic cells.
• They can replicate independently of the
host cell. The size of plasmids ranges from
a few kb to near 100 kb
• Can hold up to 10 kb fragments
• Plasmids have an origin of replication,
antibiotic resistance genes as markers,
and several unique restriction sites.
Phage Cloning Vectors
• Lambda is most common phage
• Fragments up to 23 kb can be may be accommodated
by a phage vector
Cosmid Cloning Vectors
• Fragments from 30 to 46 kb can be accommodated by a
cosmid vector.
• Cosmids combine essential elements of a plasmid and
Lambda systems.
Bacterial Artificial Chromosomes(BACs)
• BACs can hold up to 300 kbs.
• The F factor of E.coli is capable of handling large segments of DNA.
• A chloramphenicol resistance gene, and a cloning segment.
Yeast Artificial Chromosomes(YACs)
• YACs can hold up to 500 kbs.
• YACs contain: a yeast centromere, two yeast telomeres, a bacterial
origin of replication, and bacterial selectable markers
Genomic library
• Made from fragments of genomic DNA
o
Genomic DNA cut up with restriction enzymes
or randomly broken by mechanical shearing
• Fragments ligated into cloning vectors
o
Small insert
 Lambda phage
 Plasmid
o
Large insert
 BACs
How to make a genomic library
ori
total genomic DNA
ampR
genomic
DNA
restrictio
n
enzyme
anneal
and ligate
plasmid (black)
ampR
ori
ampR
same
restrictio
n
enzyme
ori
ampR
ori
ori
ampR
transform
E. coli;
select for
Amp
resistanc
e
WHAT IS cDNA?
cDNA is DNA synthesized from a mature
mRNA template in a reaction catalyzed by
the enzyme reverse transcriptase.
cDNA
strand
synthesis
WHAT IS cDNA LIBRARY?
• A cDNA library is a collection of cloned
cDNA (complementary DNA) fragments
inserted into a collection of host cells,
which together constitute some portion of
the transcriptome of the organism. cDNA
is produced from fully transcribed mRNA
found in the nucleus and therefore
contains only the expressed genes of an
organism.
WHY WE CONSTRUCT cDNA LIBRARY?
• A genomic library represents all the DNA in the genome,
whether it is expressed or not. However, very often it is
really the genes that are being expressed that are our
main target.
• Finally, if we base our library on the mRNA extracted
from the target cells, rather than on their DNA, we will be
able to focus more closely on the real target, and make
the identification of the required clones much more
efficient.
• cDNA is a more convenient way to work with the coding
sequence than mRNA because RNA is very easily
degraded by omnipresent RNases. This the main reason
cDNA is sequenced rather than mRNA.
WHAT ARE THE BASIC REAGENTS
NEED FOR cDNA LIBRARY
CONSTRUCTION?
Four basic reagents needed to produce cDNA: mRNA as template, dNTPs,
reverse transcriptase and primers.
Different types of primers:
• If the mRNA has a poly-A 3’tail,then an oligo-dt primer
can be used to prime all mRNA simultaneously.
• If you only wanted to produce cDNA from a subset of all
mRNA, then a sequence-specific primer could be used
that will only bind to one mRNA sequence.
• If you wanted to produce pieces of cDNA that were
scattered all over the mRNA, then you could use a
random primer cocktail that would produce cDNA from
all mRNAs but the cDNAs would not be full length.
Making a cDNA library
• Step 1: Isolate RNA
• RNA is purified from
tissue or cell line
• The mRNA is then
isolated away from
ribosomal and tRNAs
• Column with oligo dT
is used to bind poly A
tissue or cell
mRNA
polyA
stationary support
polyT
Step 2: Obtain cDNA
from RNA
• mRNA is treated with
the enzyme reverse
transcriptase
• The enzyme copies
sequence of mRNA into
first strand of DNA
• Another enzyme is used
to make second strand
of cDNA
Step 3: Transformation
• Double-stranded cDNA is
inserted into cloning
vector
• cDNA is ligated into
cloning vector (plasmid or
phage)
• Vector is transformed or
infected into bacteria
plasmid
E. Coli
bacteria
Screening methods for cDNA libraries
• The following methods for used to screen the
cDNA libraries, they are
1. Colony Hybridization
2. Screening expression libraries with Antibody or
other probes
Colony Hybridization
• Colony DNA is attached to
membrane
• DNA is screened with
labeled probes
• DNA is labeled with
radioactivity
• Labeled DNA is allowed to
hybridize with DNA on
membrane
• After washing, positive
hybridization spots are
identified
selected
colonies
membrane
Radioactive
probe
hybridization
X-ray film
Screening expression libraries with
Antibody or other probes
The uses of cDNA library
• There are no introns
• Useful in immunological techniques(PND
disorders)
• Identification of tissue expression between two
tissues or cell type…more..
cDNA Library vs Genomic Library
Total
Gene
Chromosomal DNA
mRNA
Reverse transcription
cDNA
Restriction digestion
Complete
gene
Smaller
Library
Larger
Library