This presentation gives a small review about the RDT based methods used generally in genetic engineering. This presentation include various images about the technique, which will help the user in understanding the concepts easily.

1. By: Mithil Shetty.

2.  Recombinant DNA Technology is the preparation of
recombinant DNA invitro by cutting the DNA
molecules and splicing together the fragments from
one or more organism.

4. Lysing
enzymes.
(lysozymes)
Cleaving
enzymes
Other
enzymes
Enzymes

5. Restriction
endonuclease
Type 1
cleave at 1000 bp away from
5’end of sequece
Type 2
cleaves within or immidate
outside their recognization
sequence
Type 3
cleaves in between immidate
vicinity of recognization site

7. Other enzymes
1. Synthesizing enzymes – RT enzyme and DNA pol.
2. Joining enzymes- T4 DNA ligase
3. Alkaline phosphatase- remove phosphate group.

8. Cloning vectors:
 The vector are the DNA molecules that can carry a
foreign DNA segments and replicate inside the host
cell.
 Types- 1. Plasmid vectors.
2. Bacteriophages.
3. Cosmids.
4. Artifical chromosome vector.
5. Transposons and virus.

9. Plasmid vectors- These are naturally occurring
plasmids.
1. pBR322 vector.

10. 2. puc18 vector

11. Bacteriophage vectors – these attack the bacteria and
insert the genomic material into it.
Eg; M13 phage vector

12.  Cosmid vector – It has been constructed by
combining certain features of plasmid and cos site of
phage lambda.
 The simplest cosmid vector contains plasmid origin of
replication, a selectable marker, suitable restriction
enzyme sites and lambda ‘cos’ site,

13. Artifical chromosome vector:
YAC vector.

14. BAC vector

16. Generalized process in RDT

17. Isolation of genomic
material
Confirmation
of isolated
DNA and its
quantification
Growing and
lysis of cells
Purification of
the DNA from
other
materials

18. 1. Growing and lysis of cell.
 Grown in an appropriate nutrient medium and then at
proper centrifugation RPM the grown cells are
separated from the medium.
 then the cells lysed by proper enzymatic method
which include the use of enzymes like lysozymes,
cellulases, or chitinases.

19. 2. Purification methods.
1. Phenol:chloform method.
2. CsCl-gradient centrifugation method.
3. Alcohol precipitation method.
4. Alakline denaturation method.
5. Column Chramotography method.
a. Size exculsion.
b. Affinity chramotography.

20. 3. Confirmation by agaros gel
electrophorosis:
Agaros gel elc. set up
Gel with separated
fragments

21. 4. Quantification.
 The concentration of a solution of nucleic acid can be
determined by measuring the absorbance at 260 nm, using
a spectrophotometer.
 An A260 of 1.0 is equivalent to a concentration of 50 g ml− 1
for double-stranded DNA, or 40 g ml− 1 for single stranded
DNA or RNA.
 If the A280 is also determined, the A260/A280 ratio
indicates if there are contaminants present, such as
residual phenol or protein.
 The A260/A280 ratio should be around 1.8 for pure DNA
and 2.0 for pure RNA preparations.

22.  In addition to spectrophotometric methods, the concentration
of DNA may be estimated by monitoring the fluorescence of
bound ethidium bromide.
 This dye binds between the DNA bases (intercalates) and
fluoresces orange when illuminated with ultraviolet (uv) light.
 By comparing the fluorescence of the sample with that of a
series of standards, an estimate of the concentration may be
obtained. This method can detect as little as 1--5 ng of DNA and
may be used when uv-absorbing contaminants make
spectrophotometric measurements impossible.
 Having determined the concentration of a solution of nucleic
acid, any amount (in theory) may be dispensed by taking the
appropriate volume of solution. In this way nanogram or
picogram amounts may be dispensed with reasonable accuracy.

23. Cutting of DNA
 This is done with the Restriction endonuclease.

24. Amplification of DNA
 By Polymerase Chain Reaction.
 Principle: When an DNA strand is subjected to high
temp. it splits into 2 strands and these SS DNA are
then converted into original double strand, by
synthezing new strand in presence of DNA Pol.
Enzyme.
 Requirements: DNA template, primars, Taq
polymerase

26. Procedure of PCR

27. Insertion of DNA into host cell or organism
A. VECTOR mediated insertion.
B. CaCl2 mediated transformation.
C. Vectorless gene transfer.
1. Microinjection.
2. Electrophoration.
3. Biolistic method.
4. Direct DNA injection.

28. Microinjection. Biolistic method

29. Selection, screening, and
analysis of recombinants
GENETIC
SELECTION
use of
chromogenic
substrates
Insertional
inactivation
Complementat
ion of defined
mutations

30. use of chromogenic substrates
The colourless compound X-gal (5-bromo-4-chloro-3-
indolyl-β-Dgalactopyranoside) is cleaved by β-galactosidase
to give galactose and an indoxyl derivative. This derivative is
in turn oxidised in air to generate the dibromo–dichloro
derivative, which is blue.

31. Insertional inactivation

32. SCREENING
Nucleic acid
probes
PCR in
screening
protocols
Immunological
screening

33. Screening clone banks by nucleic acid hybridisation

34. Immunological screening

35. PCR in screening protocols

36. ANALYSIS
OF
CLONED
GENES
Characterisation
based on mRNA
translation in
vitro
Restriction
mapping
Blotting
techniques
DNA sequencing

37. Characterisation based on mRNA translation in vitro

38. Restriction mapping

39. Blotting :
Blotting technique when used with restriction enzyme and gel electrophororsis
, can be used to identify particular region of gene in a cloned fragement of DNA
•Blotting apparatus • Southern blotting

40. An Introduction to Genetic Engineering- S. T.
Nicholl.
Principles of Gene Manipulation and Genomics
by S.B. Primrose and R.M. Twyman

41. THANK YOU