Synthesis of cDNA and BAC library

submitted to
Dr. Rashid Mahmood Rana
Submitted by
Safdar ali
14-arid-3384
Jahangir nawaz
14-arid-3064
Aminah tariq
14-arid-2966
 contents
• Introduction of cDNA
• Isolation of mRNA
• Synthesis of cDNA
• cDNA from mRNA
 Introduction to cDNA
 The term cDNA refers to complementary
DNA. cDNA is known to be synthesized, or
manufactured from an mRNA or messenger
RNA template. It is synthesized in a reaction
that is catalyzed by the reverse transcriptase
and DNA polymerase enzymes
 Essential to note is that cDNA is usually used to
clone eukaryotic genes in prokaryotes
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 cDNA is synthesize from mRNA isolated from an organism
or tissue is represented as its cDNA insertion in a plasmid or
a phage vector.
 DNA copy of RNA molecule is produced by the enzyme
reverse transcriptase(RNA dependent DNA polymerase).
 This enzyme performs similar reactions as DNA polymerase.
 Oligonucleotide primer is required which is combine with
the mRNA molecule with the poly A tail at the 3’end.
• In the prokaryotes directly DNA fragment use because in
prokaryote intron are absent.
 mRNA isolation
• In the synthesis of cDNA MATURE mRNA is
used because in pre- mature RNA introns
sequences also present
 mRNA ISOLATION
• Most eukaryotic mRNAs are polyadenylated at
their 3’ ends

5’ cap AAAAAAAAAAn

• oligo (dT) can be bound to the poly(A) tail

and used to recover the mRNA.
SYNTHESIS OF cDNA
1. Obtain mRNA. Poly(A)+mRNA is especially essential for this because
the poly A tail binds to the oligo-dT DNA.
2. Copy mRNA with the enzyme reverse transcriptase.
3. Degrade mRNA with base.
4. Make double stranded cDNA using the mRNA as template, DNA
polymerase from E.coli, and deoxyribonucleoside triphosphate as
substrates. A hairpin loop at the end of the cDNA is formed by
reverse transcriptase, may serve as primer.
5. Treat with S1 nuclease to remove the hairpin loop.
6. The resulting double stranded DNA is ligated into the cloning vector.
7. The double stranded cDNA may be used as a probe in hybridization.
 What are BAC libraries?
(Bacteria Artificial chromosome)
• During the Human Genome Project, researchers had to
find a way to reduce the entire human genome into
chunks, as it was too large to be sequenced in one go.
To do this they created a store of DNA fragments called
a BAC library. 
• What is a BAC?
• These are small pieces of bacterial DNA that can be
identified and copied within a bacterial cell.
• In general BAC clones carry inserts of DNA up to
200,000 base pairs long.
 Making a BAC library

• To make a genomic Bacterial Artificial Chromosome

(BAC) library you first have to isolate the cells
containing the DNA you want to store. For animal
and human BAC libraries the DNA normally comes
from white blood cells.?
• These isolated cells are then mixed with hot agarose,
a jelly-like substance.
• The whole mixture is poured into a mould to
produce a set of small blocks, each containing
thousands of the isolated cells.
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• The cells are then treated with enzymes to dissolve their cell
membranes and release the DNA into the agarose gel?.
• A DNA-cutting enzyme? is used to chop the DNA into pieces around
200,000 base pairs in length.
• These blocks of gel containing chopped up DNA are then inserted into
holes in a slab of agarose gel. The DNA fragments are then separated
according to size by electrophoresis.?
• On the other side of the gel a solution of ‘markers’ are inserted. These
are DNA fragments of known size which can be used to help identify
fragments of DNA of a particular size. This ensures that the BAC library
is made from DNA fragments of a particular size range. These sections
of the gel are cut out and the DNA fragments are extracted.
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• These DNA fragments are inserted into a BAC vector using an enzyme
called ligase to join the two bits of DNA together. They are now called
BAC clones.
• The BAC clones are added to bacterial cells, usually E. coli.
• The bacteria are then spread on nutrient rich plates that allow only the
bacteria that carry BAC clones to grow.
• The bacteria grow rapidly, resulting in lots of bacterial cells, each
containing a copy of the BAC clone.
• After they have grown, the bacteria are then ‘picked’ into plates of 96 or
384 so that each tube contains a single BAC clone.
• The bacteria can also be copied or frozen and kept until researchers are
ready to use the DNA for sequencing.
• A BAC library has been created.