Various technics of r dna

1. RECOMBINANT DNA TECHNOLOGY

2. Definition:- Recombinant DNA (rDNA) technology is
the technique used in genetic engineering that
involves the identification, isolation and insertion of
desired gene into a vector such as a plasmid or
bacteriophage to form a recombinant DNA molecule
and production of large quantities of that gene
fragment or product encoded by that gene.
Recombinant DNA
Technology

3. Basic principles of rDNA
technology.
• Generation of DNA fragments & selection of the desired piece of DNA.
• Insertion of the selected DNA into a cloning vector to create rDNA or
chimeric DNA.
• Introduction of the recombinant vectors into host cells.
• Multiplication & selection of clones containing the recombinant
molecules.
• Expression of the gene to produce the desired product.

4. Basic steps involved in the
process
1. Isolating genomic DNA of a donor.
2. Fragmenting this DNA using “molecular scissors” (enzymes).
3. Screening the fragments for a desired gene.
4. Inserting the fragments with desired gene into a “cloning vector.”
5. Introducing the recombinant vector into a competent host cell.
6. Culturing these cells to obtain multiple copies or clones of desired
fragments of DNA.
7. Using these copies to ‘Transform’ suitable host cells so as to
express the desired gene.

6. Tools Used in rDNA
Technology
Enzyme
1. Nucleases
2. Ligases
Vector
Host
DNA to be cloned

7. RESTRICTION
ENZYMES
A restriction enzyme (or restriction endonuclease) is an
enzyme that cuts double-stranded or single stranded DNA
at specific recognition nucleotide sequences known as
restriction sites.
Restriction enzymes recognize a specifies sequence of
nucleotides, and produce a double-stranded cut in the
DNA.
These cuts are of two types:
BLUNT ENDS.
STICKY ENDS.

8. These blunt ended fragments can be
joined to any other DNA fragment with
blunt ends.
Enzymes useful for certain types of
DNA cloning experiments
Blunt
ends

9. DNA fragments with complimentary sticky
ends can be combined to create new
molecules which allows the creation and
manipulation of DNA sequences from
different sources.
STICKY
END

11. TYPES OF RESTRICTION
ENZYMES
• Type I :-Type I restriction enzymes were the first to be
identified and are characteristic of two different strains (K-
12 and B) of E. coli.
• The recognition site is asymmetrical and is composed of
two portions – one containing 3-4 nucleotides, and
another containing 4-5 nucleotides – separated by a
spacer of about 6-8 nucleotides.
• Eg. EcoA I, EcoB, EcoK I, and CfrA I
• Type II :-These are the most commonly available and used
restriction enzymes.
• They are composed of only one subunit.
• Their recognition sites are usually undivided and
palindromic and 4-8 nucleotides in length, they recognize
and cleave DNA at the same site.

12. • Type lll :- Type III restriction enzymes) recognize two separate
non-palindromic sequences that are inversely oriented.
• They cut DNA about 20-30 base pairs after the recognition site.
• These enzymes contain more than one subunit.
• And require AdoMet and ATP cofactors for their roles in DNA
methylation and restriction
• Eg. EcoP I. Hinf III, and EcoP15 I

13. In molecular cloning a vector is a DNA molecule used as a
vehicle to artificially carry foreign genetic material into
another cell. Where it can be replicated and/ or expressed
(eg. Plasmids, Cosmids, Lambda Phages).
A vector containing foreign DNA is termed as recombinant
DNA.
Vectors used in rDNA
Technology

14. Types of
vectors

15. Plasmid
s
• Plasmids are extrachromosomal circular
DNA molecule that autonomously
replicates inside the bacterial cell.
• Cloning limit: 100 to 10,000 base pairs or
0.1-10 kilobases (kb).
• In their simplest form, plasmids contains a
bacterial origin of replication, an antibiotic
resistance gene, and at least one unique
restriction enzyme recognition site which
helps in cloning.
1. Eg. toluene and salicylic acid. Virulence plasmids,
which turn the bacterium into a pathogen.
2. Ti plasmid in Agrobacterium tumefaciens.

17. Bacteriophage
Vector
• Phage – derivatives of bacteriophage lambda; linear DNA
molecules, whose region can be replaced with foreign DNA
without disrupting its life cycle; cloning limit: 8-20 kb
• Lambda viral genome: 48.5 kb linear DNA with a 12 base
ssDNA “sticky end” at both ends; these ends are
complementary in sequence and can hybridize to each other
(this is the cos site: cohesive ends).
• Infection: lambda tail fibres adsorb to a cell surface receptor,
the tail contracts, and the DNA is injected.
• The DNA circularizes at the cos site, and lambda begins its life
cycle in the E. coli host.
• Eg. M13 phage.

18. Advantages:
• Useful for cloning large
DNA fragments (10-23
kbp)
• Inherent size selection for
large inserts
Disadvantages:
• Less easy to handle

19. Cosmi
ds
Cosmids – an extrachromosomal circular DNA
molecule that combines features of plasmids and
phage; cloning limit – 35-50 kb
Advantages:
Useful for cloning very large DNA fragments (32-47
kbp)
Inherent size selection for large inserts
Handle like plasmids
Disadvantages:
Not easy to handle very large plasmids
(~50 kbp)