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-Lecture 1

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-Lecture 1

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Knightsteel Xt
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© © All Rights Reserved
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Introduction to recombinant

DNA technology
Reference Book:
R. W. Old and S. B. Primrose, Principles of Gene Manipulation. An
Introduction to Genetic Engineering
T A Brown Gene Cloning & DNA Analysis- 6th Ed
Brooker, Robert J. Genetics : analysis & principles— 4th ed.
Overview of recombinant DNA
technology
Any cell-based cloning procedure has four essential
parts:
(i) a method for generating the DNA fragment for
cloning
(ii) a reaction that inserts that fragment into the
chosen cloning vector
(iii) a means for introducing that recombinant vector
into a host cell wherein it is replicated
(iv) a method for selecting recipient cells that have
acquired the recombinant clone

R. W. Old and S. B. Primrose, Principles of Gene Manipulation. An Introduction to Genetic Engineering


Steps in Molecular Cloning

Brooker, Robert J. Genetics : analysis & principles


Steps in Molecular Cloning-1

Brooker, Robert J. Genetics : analysis & principles


Steps in Molecular Cloning-2

X-Gal refers to the colorless compound 5-bromo-4-chloro-3-indolyl-β-d-galactoside.


IPTG is an acronym for isopropyl-β-d-thiogalactopyranoside, which is a nonmetabolizable lactose analogue that
can induce the lac promoter. Brooker, Robert J. Genetics : analysis & principles
Enzymes used in Recombinant
DNA technology
Enzymes for manipulating DNA
DNA manipulative enzymes can be grouped into
four broad classes, depending on the type of
reaction that they catalyze:
• Nucleases are enzymes that cut, shorten, or
degrade nucleic acid molecules.
• Ligases join nucleic acid molecules together.
• Polymerases make copies of molecules.
• Modifying enzymes remove or add chemical
groups.
Nucleases
Nucleases degrade DNA molecules by breaking
the phosphodiester bonds that link one
nucleotide to the next in a DNA strand. There
are two different kinds of nuclease
• Exonucleases remove nucleotides one at a
time from the end of a DNA molecule.
• Endonucleases are able to break internal
phosphodiester bonds within a DNA
molecule.
Endonuclease Vs Exonuclease

Brown, T. A. (2010). Gene cloning and DNA analysis: An introduction. Hoboken: Wiley-Blackwell.
Endonuclease examples

Brown, T. A. (2010). Gene cloning and DNA analysis: An introduction. Hoboken: Wiley-Blackwell.
Restriction Endonuclease
• Restriction enzymes (restriction
endonucleases) cut DNA at particular
sequences of bases.

• Two types:
– Enzymes that cut straight across – blunt ends
– Enzymes that cut in a staggered way – sticky ends
(more useful)
Discovery and function of restriction endonucleases

Brown, T. A. (2010). Gene cloning and DNA analysis: An introduction. Hoboken: Wiley-Blackwell.
Sticky ends
• Short stretches of single stranded DNA are complementary to
each other.

• If both ends are cut with the same enzyme, the sticky ends
will stick together by complementary base paring, forming
hydrogen bonds

• Annealing is the name given to the process by which sticky


ends form hydrogen bonds.
Restriction Endonuclease Nomenclature

Source: https://nptel.ac.in/courses/102/103/102103013/#
Types of Restriction endonucleases

Type II restriction endonucleases are important in gene


cloning.
Old and S. B. Primrose, Principles of Gene Manipulation.
Mode of action of type II REases

EcoRI

5´ ... G^A A T T C ... 3´


3´ ... C T T A A^G ... 5´
EcoRI

5´ ... G^ 3’ 5’ A A T T C ... 3´
3´ ... C T T A A 5’ 3’ ^G ... 5´
Example recognition sequences for REases
4-cutters:
AluI 5´ ... AG^CT ... 3´ blunt ends
MspI 5´ ... C^CGG ... 3´ 5’ overhang (2 bp)
6-cutters
PvuII 5´ ... CAG^CTG ... 3´ blunt ends
KpnI 5´ ... GGTAC^C ... 3´ 3’ overhang (4 bp)
8-cutters
NotI 5´ ... GC^GGCCGC ... 3´ 5’ overhang (4 bp)
Unusual sites
MwoI 5´ ... GCNNNNN^NNGC ... 3´ 3’ overhang
3´ ... CGNN^NNNNNCG ... 5´ (3 bp)
DNase I footprinting

If the DNA fragment has no


protein attached to it, a
family of labeled fragments
obtained, differing in size by
just one nucleotide each.
However, the bound protein
protected certain
phosphodiester bonds from
being cut by DNase I, thus the
fragments resulting
from cleavage within the
control sequence are absent.
Their absence shows up as a
“footprint”

Brown, T. A. (2010). Gene cloning and DNA analysis: An introduction. Hoboken: Wiley-Blackwell.
Locating a
transcription start
point by S1 nuclease
mapping.

Gene Cloning and DNA Analysis: An Introduction, 6th Edition


T. A. Brown
Exonuclease examples
The main distinction between different exonucleases lies in the
number of strands that are degraded when a double-stranded
molecule is attacked.

Brown, T. A. (2010). Gene cloning and DNA analysis: An introduction. Hoboken: Wiley-Blackwell.
Application of exonucleases
• Bal 31
– Double-stranded exonuclease, operates in a time-dependent
manner
– Degrades both 5’ and 3’ ends of DNA
– Useful for generating deletion sets, get bigger deletions with
longer incubations

• Exonuclease III
– has 3’→5’ exonuclease activity in a double stranded DNA.
– optimal activity with blunt ended sequences or sequences
with 5’ overhang.
– Useful for
• Site-directed mutagenesis
• Preparation of single-stranded DNA for dideoxy sequencing
• Preparation of nested deletions in double-stranded DNA

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