Recombinent Dna Technology: Gpat-Microbiology
Recombinent Dna Technology: Gpat-Microbiology
Recombinent Dna Technology: Gpat-Microbiology
GPAT-MICROBIOLOGY
RECOMBINENT DNA TECHNOLOGY
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11. DNA ligase join the DNA fragments by forming a phosphodiester bond between the
phosphate group of 5’ carbon of one deoxyribose with hydroxyl group 3’ carbon of
another deoxyribose.
12. Alkaline phosphates are an enzyme involved in the removal of Phosphate group.
13. Polynucleotide kinas involved in the addition of phosphate group.
14. Microorganism are preferred as the host cell because they multiply faster compared to
cell of higher organism ( Plant and Animal)
15. Host of choice- Escherichia coli ( Gram Negative)
16. Bacillus subtitles is a rod shaped non pathogenic bacterium, it has been used as host in
industry for the production of enzyme, antibiotic, insecticide etc.
17. Eukaryotic host- They are preferred to produce human protein because they are more
suitable. Example- Yeat, Saccharomyces cerevisiae.
18. List of important host and their example-
S.N. Name Example
01 (Prokaryotes) Bacteria Ecolli, Bacillus, Subtillis, Streptomyces.
02 (Eukaryotes) Fungi Saccharomyces Cerevisiae, Aspergillus
Nidulasns
03 Animal Insect Cell, Oocyte, Mammilian Cell
04 Plants Protoplast, Intact Cell, Whole Palnt
19. Vector- They is DNA molecule which can “carry a Foreign DNA fragments to be cloned”
They are self replicating in an appropriate host cell.
Example- Plasmid, Bacteriophage, cosmid, phasmids.
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20. Plasmid- They extra chromosomal double standard circular, self replicating DNA
molecule. Almost all bacteria have plasmid contains low number (01-04) to the high
number (10-100 per cell)
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21. Bacteriophage- bacteriophage is the virus that replicate within bacteria.
22. Cosmid- They are the vector possessing the characteristic of both plasmid and
bacteriophage lemda.
23. Artificial Chromosome Vector- Developed in 1997 by H. Willard human artificial
chromosome is a synthetically produced vector DNA, possessing the characteristic of
human chromosome.
24. Shuttle Vector- the Plasmid vector that are specifically designed to replicate in two
different hosts. Ecoli and strptomyces are referred to as shuttle vector.
25. Method of gene transfer-
(1) Transformation
(2) Conjugation
Solution
(3) Electroporation www.facebook.com/pharmavideo/
(4) Lipofection
(4) Direct Transfer
26. Transformation- It is the method of introducing foreign DNA into bacterial cell. The
update of plasmid DNA by Ecolli is carried out in ice cold CaCl2 (0-5oC) and a
subsequent heat shock (37-45oC) for about 90 Second.
27. Transformation Efficiency- it refers to the number of Transformation per microorganism
of added DNA,
Mechanism- It is believed that CaCl2 affects the cell wall and break at localized region
and is also responsible for binding DNA to cell surface. Large size DNA is less efficient
in transforming.
28. Conjugation- it is a natural microbial recombination process. During conjugation two live
bacteria come together join by cytoplasmic bridge and transfer single slandered DNA into
cell.
29. Electroporation- it is based on the principle that high voltage electric pulse can induce
cell plasma membrane to fuse, thus Electroporation is a technique involve electric field
mediated membrane permiabilization. Electric shock can also be inducing cellular intake
of exogenous DNA from the suspending solution.
The cell is placed in a solution containing DNA to move across the host cell and thus the
Electroporation process gets completed.
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30. Liposome mediated gene transfer- liposome are arcular lipid molecule which have an
aqueous interior that can carry nucleic acid. On treatment of DNA fragments with
liposome the DNA piece get encapsulated inside liposome. This liposome can adhere to
cell membrane and fuse with them to transfer DNA fragments. Thus DNA enters the cell
and then to the nucleuses. The positively charged liposome very efficiently complex with
DNA, bind to cell and transfer DNA rapidly.
31. Transduction- Sometime the foreign DNA can be packed inside animal virus. This virus
can naturally infect the cell and induce the DNA into host cell. This type of transfer is
known as transduction.
32. Microinjection
36. Isolate protoplast are expressed to a mixture of 5.5% NAHCO3 in 10% sucrose solution.
37. PEG treatment is done for-
(1) Result in reproducible high frequency of heterokaryon formation.
(2) Low toxicity in cell
(3) Reduced the formation of binucleotyte heterokaryon
(4) PEG induced fusion is non specific and therefore can be used for a wide range of
plant.
38. Those substance which induced fusion are known as – Fusogens
39. Example of fusogens are- PEG, high PH, high Ca2+ cintent.
40. High PH and high Ca2+ ions neutralize the surface charge.
41. High temperature helps in the membrane fusion.
42. Monoclonal antibody is used for the production of antibody synthetically in the
biotechnology laboratory.
43. Antibody or immunoglobulin is protein molecule produced by Beta lymphocyte.
44. Monoclonal antibody is antibody that is desired against a specific antigen determinant.
4
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Solution
45. Specific antigen determine site is known as Epitope www.facebook.com/pharmavideo/
5
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54. Biotechnology is defined as a use of living organism to produce product, which is
beneficial for mankind. Solution
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55. Milestone in the field of Biotechnology-
S.N Year Scientist Milestone
1962 Arber
1968 Messelson Site specific reorganization and cleavage of
01 1970 Smith DNA by Restriction endonuclease.
1971 Nathans
02 1966 Nirenberg, leader, Determination of genetic code
Khorana
03 1967 Gellert Determination of genetic code
04 1970 Temin and Mizutani Reverse Transcription
Baltiomore
05 1971-72 Boyer, Cohen and beg DNA Cloning Technique
06 1975 Milstein & Kohler Hybridoma Technology
04 1977 Sanger, Gilbert DNA sequencing technology
05 1981 Anti-C3d Bioclo0ne US Approval of 1st Diagnostic kit based on
ortho Diagnostic monoclonal technology.
06 1982 Genetech and Eli Lilly US Approval Od 1st Ethical Pharmaceutical
produced by using rDNA technology.
Human Insulin (Humulin)
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6
MICROBIOLOGY