discuss about various tools used in genetic engineering or recombinant DNA technology.

1. Tools used in Genetic
Engineering
PREPARED BY:
MS. DIPTEE GUPTA
ASSISTANT PROFESSOR
KRISHNA INSTITUTE OF PHARMACY & SCIENCES,
KANPUR
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2. Cloning Vector
Vectors are DNA molecules which can carry a foreign DNA fragment to be
cloned. They are self replicating in an appropriate host cell.
Any extra-chromosomal small genome is used as a vector.
Eg: Plasmid, Bacteriophage, Cosmid, Yeast, Shuttle, Expression vector etc.
Characteristics:
•Small in size
•Have a single restriction endonuclease site
•Have an origin of replication (Ori)
•Have 1-2 suitable marker genes, allow easy selection of transformed cells.
•Should easily isolated from cells.
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3. 1. Plasmid vector
These are extra-choromosomal, double stranded, circular, self
replicating DNA molecules present in bacterial cell.
They are widely used as as cloning vehicles.
Almost all the bacteria have plasmids containing a low copy of number
(1-4 per cells) or a high copy number (10 -100per cells).
The size of plasmid varies from 1-500 kbp (kilo base pair).
Eg: pBR322, pBR325, pUC19, pBR329, pMB9, pRK2501
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4. pBR322 Vector:
It is one of the artificial vector & most widely used plasmid from Escherichia coli
plasmid.
It have 4362 basepair (bp) in length.
In the name of pBR322 P stands for Plasmid, BR stands for scientist name
Bolivar & Rodriguez.
The structure of pBR322 have:
Origin of replication (Ori)
Restriction enzyme site: almost 20 different restriction sites present in which 11
sites present in Tetracycline resistant region while 9 present in Ampicillin resistant
region. Eg: Bam HI, HIND III, EcoR IV
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5. Genetic markers: 2 selectable markers present –
Ampicillin resistant site i.e the ampicillin gene codes for β-lactamase, which can be
used for screening microorganisms when a foreign DNA is being inserted in the
plasmid.
Tetracycline resistance site – this gene degrades the antibiotic tetracycline and can be
used for screening microorganisms.
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6. 2. Bacteriophage vector:
These are those virus which can replicate within
bacteria.
These vectors can accept short fragments of
foreign DNA but they can carry larger segment
than plasmid.
Bacteriophage of E.coli: λ, Bacteriophage
M13 & Fd.
These vectors can carry larger segment over
25kbp as compare to plasmid vector.
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7. 3. Cosmid vectors: (cos site + Plasmid)
•These are special type hybrid vectors that carry characteristics of both
plasmid and bacteriophage vectors (λ).
•They can carry upto 45 kb long DNA segment and can replicate as plasmid
if they have suitable Ori.
•These can also packed in Phage capsid that allow the foreign gene to be
transferred into cell by transduction.
Some artificial chromosome vectors:
Human artificial chromosome (HAC)
Yeast artificial chromosome (YAC)
Bacterial artificial chromosome (BAC)
Shuttle vector
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8. Yeast artificial chromosome (YAC):
These are expression vectors that allow transcription and translation of
inserted section of DNA.
But these vectors usually produce chimeric effect, that make them less stable
as compare to BAC.
Shuttle vector:
It is a plasmid vector but designed to replicate in 2 host cells such as E.coli &
Streptomyces species. Generally the Ori of 2 vectors, combine in one vector
to form a shuttle vector.
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9. Restriction Endonuclease
These are one of the most important group of enzymes used for
manipulation of DNA.
These are bacterial enzyme that can cut/split at specific nucleotide site
(Palindromic sequence) and also make cut on plasmid and desired
DNA.
The target site for cutting may vary from one enzyme to another enzyme.
These are also known as biological knives or biological scissors and
belong to class of enzymes called Nucleases.
HIND II first restriction enzyme to be isolated.
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10. Nomenclature: named by a standard procedure—
First letter- indicate genus
Next 2nd and 3rd letter- indicate species
4th letter – strain of organisms
Roman number- indicate the order isolated from strain
Eg; Eco RI Escherichia (E), Coli (C), Strain – RY 13 (R), first
endonuclease (I) to be isolated from strain
Hind III Haemophilus (H), influenzae (in), Strain – Rd (d) , third
endonuclease enzyme
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11. Types :
There are 3 main types of Restriction Endonuclease- Type I , Type II, Typer III.
These enzymes differ from each other in their mode of action.
Type I Type II Type III
Consist of 3 different
subunits
2 Identical subunit 2 different subunit
Require ATP, Mg++, S-
adenosyl methionine
for restriction
Require Mg++ for
restriction
Require ATP, Mg++, S-
adenosyl
Cleaves DNA at
random site upto
1000bp away from
recognition sequence
Cleaves within
recognition sequence
Cleaves DNA about
25bp from recognition
sequence
Not used in rDNA Used in rDNA
technology
Not used
Eg: Eco K I, Eco BI Eg; Eco RI, Alu I Eg: Pst I, Hinf III
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12. Mechanism of Restriction Endonuclease
These enzymes cut / cleave the DNA molecule in 2 different ways-
1.Blunt end / Flush end- when enzyme cleave both strand of DNA at
the same point within recognition sequence, blunt ends are generated.
Eg: Puv II, Hae III
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13. 2. Sticky end:
In this , 2 strands of DNA are cut at different points, it generate
protruding ends i.e. one strands of double helix extends a few base
beyond the other strand.
Eg; Eco RI
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14. DNA Ligase:
It is a type of enzyme that facilitate the joining of DNA strands together
by catalyzing the formation of phosphodiester bond.
This phosphodiester bond is formed between phosphate group of 5`
carbon of one deoxyribose with the –OH group at 3` carbon of another
deoxyribose.
Originally isolated from viruses but occur in E.coli & eukaryotic cells.
Types:
T4 DNA ligase: require ATP as cofactor & have the ability to join the
blunt end of DNA fragments.
E.Coli DNA ligase: require NAD+ as a cofactor & have the ability to
join the sticky ends produced by restriction enzymes
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15. DNA ligase enzymes participated in cellular DNA repair process and
also in DNA replication process.
It have extensive use in molecular biology laboratories for recombinant
DNA experiments.
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