Recombinant DNA technology and other methods.pdf

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

Recombinant DNA is the general name for a piece of DNA that has been created by combining two or more fragments from different sources. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, differing only in the nucleotide sequence. Recombinant DNA molecules are sometimes called chimeric DNA because they can be made of material from two different species like the mythical chimera. rDNA technology uses palindromic sequences and leads to the production of sticky and blunt ends.

The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA can be joined to bacterial DNA, or human DNA can be joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature can be created by the chemical synthesis of DNA and incorporated into recombinant DNA molecules. Using recombinant DNA technology and synthetic DNA, any DNA sequence can be created and introduced into living organisms.

Proteins that can result from the expression of recombinant DNA within living cells are termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein is not necessarily produced.[1] Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring by foreign coding sequences.[2]

Recombinant DNA differs from genetic recombination in that the former results from artificial methods while the latter is a normal biological process that results in the remixing of existing DNA sequences in essentially all organisms.

Production

Main article: Molecular cloning
Molecular cloning is the laboratory process used to produce recombinant DNA.[3][4][5][6] It is one of two most widely used methods, along with polymerase chain reaction (PCR), used to direct the replication of any specific DNA sequence chosen by the experimentalist. There are two fundamental differences between the methods. One is that molecular cloning involves replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free of living cells. The other difference is that cloning involves cutting and pasting DNA sequences, while PCR amplifies by copying an existing sequence.

Formation of recombinant DNA requires a cloning vector, a DNA molecule that replicates within a living cell. Vectors are generally derived from plasmids or viruses, and represent relatively small segments of DNA that contain necessary genetic signals for replication, as well as additional elements for convenience in inserting foreign DNA, identifying cells that contain recombinant DNA etc etc.....................

2. It occurs in following stages
Generation of DNA fragments & selection of
the desired piece of DNA(e.g. a human gene).
Insertion of the selected DNA into a cloning
vector (e.g. a plasmid) to create a
recombinant DNA or chimericDNA.

3. Introduction of the recombinant vectors into
host cells (e.g. bacteria).
Multiplication & selection of clones
containing the recombinant molecules.
Expression of the gene to produce the
desired product.

5. 1.Molecular tools of genetic engineering.
2.Host cells-the factories of cloning.
3.Vectors-the cloning vehicles.
4.Methods of gene transfer.
5.Gene cloning strategies.

6. Restriction endonucleases -DNA cutting
enzymes:
Restriction endonucleases are one of the most
important groups of enzymes for the
manipulation of DNA.
These are the bacterial enzymes that can
cut/split DNA (from any source) at specific sites.

7. They were first discovered in E.colirestricting
the replication of bacteriophages, by cutting
the viral DNA (The host E. coli DNA is
protected from cleavage by addition of
methyl groups).
Thus, the enzymes that restrict the viral
replication are known as restriction enzymes
or restriction endonucleases.

8. Recognition sequence is the site where the
DNA is cut by a restriction endonuclease.
Restriction endonucleases can specifically
recognize DNA with a particular sequence of
4-8 nucleotides & cleave.
Cleavage patterns: Majority of restriction
endonucleases (particularly type II) cut DNA
at defined sites within recognition sequence.

9. The cut DNA fragments by restriction
endonucleases may have mostly sticky ends
(cohesive ends) or blunt ends.
DNA fragments with sticky ends are useful for
recombinant DNA experiments.
This is because the single-stranded sticky DNA
ends can easily pair with any other DNA
fragment having complementary sticky ends.

10. The cut DNA fragments are covalently joined
together by DNA ligases.
These enzymes were originally isolated from
viruses.
They also occur in E.coli& eukaryotic cells.
DNA ligasesactively participate in cellular
DNA repair process.

11. The hosts are the living systems or cells in
which the carrier of recombinant DNA
molecule or vector can be propagated.
There are different types of host cells-
prokaryotic(bacteria) & eukaryotic(fungi,
animals & plants).

12. Host cells, besides effectively incorporating
the vector's genetic material, must be
conveniently cultivated in the laboratory to
collect the products.
Microorganisms are preferred as host cells,
since they multiply faster compared to cells
of higher organisms (plants or animals).

13. Escherichia coli:
Escherichia coli was the first organism used
in the DNA technology & continues to be the
host of choice by many workers.
The major drawback is that E. coli (or even
other prokaryotic organisms)cannot
perform post-translational modifications.
Bacillus subtilisas an alternative to E.coli.

14. The most commonly used eukaryotic organism
is the yeast, Saccharomycescerevisiae.
Certain complex proteins which cannot be
synthesized by bacteria can be produced by
mammalian cells e.g. tissue plasminogen
activator.
The mammalian cells possess the machinery to
modify the protein to the active form (post-
translational modifications).

15. Vectors are the DNA molecules, which can
carry a foreign DNA fragment to be cloned.
They are self-replicating in an appropriate
host cell.
The most important vectors are plasmids,
bacteriophages, cosmids& artificial
chromosome vectors.

16. Plasmids are extrachromosomal, double-
stranded, circular, self-replicating DNA
molecules.
Almost all bacteria have plasmids.
Size of plasmids varies from 1 to 500 kb.
Plasmids contribute to about 0.5 to 5.0% of
total DNA of bacteria.

18. pBR322 has a DNA sequence of 4,361 bp.
It carries genes resistance for ampicillin
(Amp1) & tetracycline (Tel1) that serve as
markers for the identification of clones
carrying plasmids.
The plasmid has unique recognition sites for
the action of restriction endonucleases-EcoRl,
Hindlll, BamHl, Sall& Pstll

19. The other plasmids employed as cloning
vectors include pUC19 (2,686 bp, with
ampicillinresistance gene) & derivatives of
pBR322-pBR325, pBR328 & pBR329.

20. Bacteriophagesor phages are the viruses
that replicate within the bacteria.
In case of certain phages, their DNA gets
incorporated into the bacterial chromosome
& remains there permanently.
Phage vectors can accept short fragments of
foreign DNA into their genomes.

21. Phages can take up larger DNA segments
than plasmids.
Phage vectors are preferred for working
with genomes of human cells.
The most commonly used phages are
bacteriophageλ(phage λ) & bacteriophage
(phage M13).

22. Cosmidsare victors possessing the characteristics of
both plasmid & phage λ.
Cosmidscan be constructed by adding a fragment of
phage λDNA including Cossite, to plasmids.
A foreign DNA (about 40 kb) can be inserted into
cosmidDNA .
The recombinant DNA, formed can be packed as
phages & injected into E.coli.
Inside host cell, cosmidsbehave like plasmids &
replicate & can carry larger fragments of foreign DNA

23. Human artificial chromosome (HAC):
Artificial chromosome is a synthetically
produced vector DNA, possessing the
characteristics of human chromosome.
HAC may be considered as a self-replicating
microchromosomewith a size ranging from
1/10
th
to 1/5
th
of a human chromosome.
It can carry long human genes.

24. Yeast artificial chromosome (YAC) is a
synthetic DNA that can accept large
fragments of foreign DNA(particularly
human DNA).
It is possible to clone large DNA pieces by
using YAC.

25. Construction of BACs is based on one F-
plasmid which is larger than the other
plasmids used as cloning vectors.
BACs can accept DNA inserts of around 300 kb.

26. Transformation:
Transformation is the method of introducing
foreign DNA into bacterial cells (e.g. E.coli).
Uptake of plasmid DNA by E.coliis carried
out in ice-cold CaCl2 (0-5˚C) & a subsequent
heat shock (37-45˚C for about 90 sec).

27. A natural microbial recombination process.
During conjugation, two live bacteria (a
donor & a recipient) come together, join by
cytoplasmicbridges & transfer single
stranded DNA (from donor to recipient).
In side recipient cell, new DNA may integrate
with the chromosome or may remain free.

28. It is a technique involving electric field
mediated membrane permeabiIization.
Electric shocks can also induce cellular uptake
of exogenous DNA (believed to be via the
pores formed by electric pulses) from the
suspending solution.
It is a simple & rapid technique for
introducing genes into cells.

29. Liposomes are circular lipid molecules, which
have an aqueous interior that can carry
nucleic acids.
Several techniques have been developed to
encapsulate DNA in liposomes.
The liposome mediated gene transfer is
referred to as lipofection.

30. Treatment of DNA fragment with liposomes,
DNA pieces get encapsulated inside liposomes.
These liposomes can adhere to cell membranes
& fuse with them to transfer DNA fragments.
The DNA enters the cell & to the nucleus.
Positively charged liposomes efficiently
complex with DNA, bind to cells & transfer DNA

31. It is possible to directly transfer the DNA into
the cell nucleus.
Microinjection & particle bombardment are
the two techniques used for this purpose.

32. A clone refers to a group of organisms,
cells, molecules or other objects, arising
from a single individual.

33. Generation of DNA fragments
Insertion into a cloning vector
Introduction into host cells
Selection or screening
RE-digestion, cDNAsynthesis, PCR,
chemical synthesis
Ligation of blunt ends, homopolymer
tailing, linker molecules
Transformation, transfection,
tradsduction
Hybridization, PCR, immunochemical methods, protein-
protein interactions, functional complementation

34. Textbook of Biochemistry –U Satyanarayana