it includes tools ,necessary of recombinantDNA technology,overview & explanation of recDNA process

1. D ARUNKUMAR
Department of Biotechnology
B.Tech IIIrd year

2. Basics of Rec DNA Technology
Steps involved in Rec DNA Technology
Tools required for it
Advantages

3.  Gene cloning molecular cloning DNA cloning
 Mutliplicaton of same copies of DNA again and again

4. Prokaryotes system – smaller protein
Eukaryotes system – larger protein
(To fulfill the smaller protein requirements)
yeasts bacteria
Sacchromyces cerevisiae
Pichia pastoris
Hansenula polymorpha
High yield , low cost , >50KDa
Thermal tolerant ,uptake of unusual
carbon source
E coli
Provide s-s proteins ,easier in PTM

5. 1) Selection and isolation of DNA
2) Selection of suitable vector
3) Introduction of Gene of interest to vector
4) Selection and Insertion in host cells
5) Expression and multiplication of recDNA

6. Selection : To find a part of DNA which responsible
for expression of protein system
For Insulin human insulin gene is taken
Isolation : To isolate the expression system by
enzymatically (Restriction endonuclease )
They are also termed as DNA insert , foreign DNA
,target DNA ,clone DNA
It can be done by Gel electrophoresis

7. They are vehicle of the rec DNA technology which
carry the information of DNA over a generation
They are self replicating in nature
Commonly Plasmids and rarely bacteriophage are
used
Vector cannot be created

8. To join the gene of interest and vector DNA by
enzymatically
Ligase is a most commonly used enzyme in rec
DNA

9. Slight distruption of
cell wall and the
recombinant molecule
was inserted into a
suitable host by
enzymatically
Diferent host system
were used

12. I. Enzymes
II. Cloning vector
III. Foreign DNA
IV. Host organisms
V. Linker and adapter sequences

13.  Necessary tools in RDNA
 Commonly used enzymes are
Endonuclease
Exonuclease
Ligase
ENDONUCLEASE
To cut DNA at a specific site (recognition site
/recognition sequence /restriction site /target
site )
In 1962 W.ARBER
Found that some of
enzyme present in
Bacteria which can
degrade DNA
By inserting phage
into a E.coli , found
the restricting the
growth of PHAGE
In 1970 MESELSON
&YUAN were isolate
true endonuclease

14. Types
i) Type I
ii) Type II
iii) Type III
TYPE I RESTRICTION ENDONUCLEASE
cleave one strand of DNA at a specific site
Requires Mg2+ ions & ATP for functioning

15. Most stable
cleave both strand at a definite length
requires Mg2+ ions & not ATP
So, advantageous over a TYPE I
Cuts between a palindrome sequence
with rotational symmentry

16. Not used in a gene cloning / rec DNA Technology
Its an intermediate between TYPE I & TYPE II

17. By Two ways
Blunt cut
i)Cleave both double strand at the same point
Staggered cut
ii) cleave the double strand at different point
protruding ends , formation of sticky ends /
cohesive ends ( to pair easily )

18. Removes a part of a
nucleotide
Can remove either
ends(5’ /3’ ) of a DNA
Never produce internal
cuts
Join two fragments
By synthesizing the
phosphodiester bonds
Called as a molecular
glue

19. Capable to replicate in host organism
Called as a cloning vehicle / Earner DNA
Developed from Bacillus ,Pseudomonas ,Agrobacterium ,Yeast &
Fungi
Diff. vector system were used
Plasmids , bacteriophage , cosmids , Phasmids
For a good cloning vector have single site for cutting
They should perform glycosylation
Express more quantity of protein in terms of quality & quantity
Should have a ori of replication

20. Lack of post translational modifications
Mutations may arise whenever improper addition of
segments
In wwPDP E.coli is used as vector for 23,462
processing

21. They carries the recDNA and multiplies within a cell and
involves the cell division
As a result of cell division large number of recombinated cell
are produced
 which inturn results in the expression of proteins
They are easy to transform
Easy to replicate
Should interfere against replication of recDNA in host cells

22.  DNA which have to be cloned is called DNA interest or foreign DNA or target DNA
 They are responsible for the expression of protein in recDNA technology
 They are made up of certain sequence of nucleotides which are responsible
 They may be viral /plant /animal /bacterial DNA part