Gene Cloning.ppt

DNA cloning is the process of making multiple, identical copies of a particular piece of DNA.
DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene.
In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid.
The plasmid is introduced into bacteria via a process called transformation, and bacteria carrying the plasmid are selected using antibiotics.
Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express the gene and make protein.
BASIC STEPS OF GENE CLONING are :
1. Cutting and pasting DNA and Vectors
2. Bacterial transformation and selection
3.Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it
Basic Requirements for Gene Cloning:
Cloning Vectors
Isolation Procedure for DNA fragment and Vectors
Appropriate Enzyme for Purified DNA manipulation
Host Cells
Agar with Antibiotics
Vectors for Gene Cloning: Plasmid and Bacteriophage :
Characteristic of a Vector for Gene cloning
It must be able to replicate with in the host cells
It needs to be small , Ideally less than 10 kb in size

Two kind of DNA molecule that satisfy the above criteria can be found in bacterial cells, namely Plasmid and Bacteriophage chromosomes

Plasmid:Is a circular molecule of DNA that lead un independent existence in the bacteria cells.
Bacteriophages, or phages are viruses that specifically
infect bacteria.
Characteristic of a Vector for Gene cloning
It must be able to replicate with in the host cells
It needs to be small , Ideally less than 10 kb in size

Two kind of DNA molecule that satisfy the above criteria can be found in bacterial cells, namely Plasmid and Bacteriophage chromosomes

Plasmid:Is a circular molecule of DNA that lead un independent existence in the bacteria cells.
Bacteriophages, or phages are viruses that specifically
infect bacteria.
Characteristic of a Vector for Gene cloning
It must be able to replicate with in the host cells
It needs to be small , Ideally less than 10 kb in size

Two kind of DNA molecule that satisfy the above criteria can be found in bacterial cells, namely Plasmid and Bacteriophage chromosomes

Plasmid:Is a circular molecule of DNA that lead un independent existence in the bacteria cells.
Bacteriophages, or phages are viruses that specifically
infect bacteria.
The procedure for total DNA preparation from a culture of bacterial cells can be divided into four stages (Figure 3.1):
A culture of bacteria is grown and then harvested.
The cells are broken open to release their contents.
This cell extract is treated to remove all components except the DNA.
The resulting DNA solution is concentrated
Treatment with EDTA and lysozyme is carried out in the presence
of sucrose, which prevents the cells from bursting immediately.

1. Gene Cloning
Solomn A.

2. What is Gene Cloning?
DNA cloning is the process of making multiple, identical
copies of a particular piece of DNA.
It is an insertion of DNA fragment to a vector molecule
and then transferring to the host cells( bacteria) .

3. BASIC STEPS OF GENE CLONING
The basic steps are:
1.CuttingandpastingDNAandVectors
2.Bacterialtransformationandselection
3.Growuplotsofplasmid-carryingbacteria
andusethemas"factories"tomakethe
protein.Harvesttheproteinfromthebacteria
andpurifyit

4. Basic Requirements for Gene Cloning
Cloning Vectors
Isolation Procedure for DNA fragment and
Vectors
Appropriate Enzyme for Purified DNA
manipulation
Host Cells
Agar with Antibiotics

5. Vectors for Gene Cloning: Plasmid and Bacteriophage
CharacteristicofaVectorforGenecloning
Itmustbeabletoreplicatewithinthehostcells
Itneedstobesmall,Ideallylessthan10kbinsize
TwokindofDNAmoleculethatsatisfytheabovecriteriacanbe
foundinbacterialcells,namelyPlasmidandBacteriophage
chromosomes
Plasmid:IsacircularmoleculeofDNAthatleadunindependent
existenceinthebacteriacells.
Bacteriophages,orphagesarevirusesthatspecifically
infectbacteria.

6. Plasmid Vectors

7. Representative plasmids

8. Bacteriophage

9. 1. Purification of fragment DNA and vectors
The procedure for total DNA preparation from a culture of
bacterial cells can be divided into four stages (Figure 3.1):
1)A culture of bacteria is grown and then harvested.
2)The cells are broken open to release their contents.
3)This cell extract is treated to remove all components
except the DNA.
4)The resulting DNA solution is concentrated.

10. Preparation of total cell DNA

11. The composition of two typical media for the growth of
bacterial cells

12. Harvesting by centrifugation

13. Preparation of cell extract

14. Approaches of DNA purification

15. Plasmid purification by the Alkaline denaturation method

16. Bacteriophage vector preparation

17. Collection of phage particles by PEG

18. 2. Manipulation of purified DNA
DNAmanipulativeenzymescanbegroupedinto
fourbroadclasses,dependingonthetypeof
reactionthattheycatalyse:
1.Nucleasesareenzymesthatcut,shorten,or
degradenucleicacidmolecules.
2.Ligasesjoinnucleicacidmoleculestogether.
3.Polymerasesmakecopiesofmolecules.
4.Modifyingenzymesremoveoraddchemical
groups.

19. Nuclease
Nucleases degrade DNA molecules by breaking the phosphodiester
bondsthat link one nucleotide to the next in a DNA strand.
There are two different kinds of nuclease
1.Exonucleasesremove nucleotides one at a time from the end of a
DNA molecule.
Bal31
ExonuleaseIII
2. Endonucleasesare able to break internal phosphodiesterbonds
within a DNA molecule.
S1 nuclease
DNease
Restriction Endonuclease

20. Exonuclease and endonuclease enzyme

22. Exonulease

23. Endonuclease

24. Ligase( DNA Ligase)

25. DNA Polymerases

26. DNA Modifying Enzyme

27. Cutting Plasmid vector molecule and Target gene

28. Cutting Phage vector molecule and Target gene

29. Construction of Recombinant DNA

30. 4. Transformation: The uptake of DNA by bacterial cells

31. The product of ligation

33. Nutrient Agar

34. Preparation of Recombinant DNA

35. Gene cloning by using plasmid vector

36. Gene cloning by using bacteriophage

37. Uses of DNA cloning
DNAmoleculesbuiltthroughcloningtechniquesare
usedformanypurposesinmolecularbiology.
Ashortlistofexamplesincludes:
Biopharmaceuticals
Genetherapy
Geneanalysis

38. END
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