The document discusses DNA libraries, specifically genomic and complementary DNA (cDNA) libraries, detailing their creation and characteristics. Genomic libraries contain copies of every DNA sequence in an organism's genome, while cDNA libraries contain sequences corresponding to expressed mRNA. It also covers the sequencing of cloned DNA fragments, automated DNA sequencing methods, and various enzymes used in molecular biology.

1. DNA LIBRARY
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. DNA libraries or Gene Library
 A DNA library is a collection of cloned restriction
fragments of the DNA of an organism.
 Two kinds of libraries will be discussed: genomic
libraries and complementary DNA (cDNA) libraries.
 Genomic libraries ideally contain a copy of every
DNA nucleotide sequence in the genome.
 In contrast, cDNA libraries contain those DNA
sequences that appear as mRNA molecules, and these
differ from one cell type to another.

3. 1.Genomic DNA libraries:
 A genomic library is the collection of
fragments of dsDNA obtained by digestion
of the total DNA of the organism with a
restriction endonuclease and subsequent
ligation to an appropriate vector.
 The recombinant DNA molecules are
replicated within host bacteria.

4. Contd…
 The amplified DNA fragments represent
the entire genome of the organism and are
called a genomic library.
 Note: Cloned cDNA lack introns and the control
regions of the genes, whereas these are present in
genomic libraries.

6. 2.cDNA libraries:
 If a gene of interest is expressed at a very high
level in a particular tissue, it is likely that the
messenger RNA (mRNA) corresponding to that
gene is also present at high concentrations in the
cell.
For example, reticulocyte mRNA is composed largely
of molecules encoding the α-globin and β-globin chains
of hemoglobin.

7. Contd…
 This mRNA can be used as a template to make a
complementary dsDNA (cDNA) molecule using
the enzyme reverse transcriptase
 The resulting cDNA is thus a double-stranded
copy of mRNA.
 cDNA can be amplified by cloning or by the
polymerase chain reaction.

8. Fig : Synthesis of cDNA from mRNA using reverse transcriptase.

9. Sequencing of cloned DNA fragments:
 The base sequence of DNA fragments that have
been cloned and purified can be determined in the
laboratory.
 The original procedure for this purpose was the
Sanger dideoxy method illustrated in Figure.
 The ssDNA to be sequenced is used as the template
for DNA synthesis by DNA polymerase.
 A radioactive primer complementary to the 3′-end
of the target DNA is added, along with the four
deoxyribonucleoside triphosphates (dNTP).

10. Contd…..
 The sample is divided into four reaction tubes,
and a small amount of one of the four
dideoxyribonucleoside triphosphates (ddNTP)
is added to each tube.
 Because it contains no 3′-hydroxyl group,
incorporation of a ddNTP into a newly
synthesized strand terminates its elongation at
that point.

11. Contd…..
 The products of this reaction then consist of a
mixture of DNA strands of different lengths,
each terminating at a specific base.
 Separation of the various DNA products by size
using polyacrylamide gel electrophoresis,
followed by autoradiography, yields a pattern of
bands from which the DNA base sequence can
be read.

14. Automated DNA sequencing:
 Modern automated DNA sequencing systems
designed to generate over half a million
bases of sequence per day use sequencing
gels contained in multiple capillary tubes
(rather than in a slab gel format).
 Preparation and loading of the samples onto
the capillary gels is carried out by robots and
data analysis is also automated.

15. Alkaline phosphatase
Removes phosphate groups from 5' ends of
DNA (prevents unwanted re-ligation of cut
DNA)
DNA ligase
Joins compatible ends of DNA fragments
(blunt/blunt or complementary cohesive
ends). Uses ATP
DNA polymerase I
Synthesises DNA complementary to a DNA
template in the 5'-to-3'direction. Starts from
an oligonucleotide primer with a 3' OH end
Exonuclease III
Digests nucleotides progressiviely from a
DNA strand in the 3' -to-5' direction
Polynucleotide kinase
Adds a phosphate group to the 5' end of
double- or single-stranded DNA or RNA. Uses
ATP
RNase A Nuclease which digests RNA, not DNA
Taq DNA polymerase
Heat-stable DNA polymerase isolated from a
thermostable microbe (Thermus aquaticus)
ENZYMES USED IN
MOLECULAR BIOLOGY

16. ENZYMES USED IN
MOLECULAR BIOLOGY