cloning and sub-cloning

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1 Presented by: Sandhya Talla M.Pharm (Pharmacology) Cloning and Sub cloning

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Presented by:Sandhya Talla

M.Pharm (Pharmacology)

Cloning and Sub cloning

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Clone:- A collection of cells or organisms that are genetically identical. An identical genetic copy of an organism - animal/plant/ human being.

Cloning:- Cloning is the creation of an organism that is an exact genetic copy of another. This means that every single bit of DNA is the same between the two!

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History

In 1928, German Dr. Hans Spemann performed the first nuclear transfer experiment with salamander embryos, where he transferred the nucleus of a salamander embryonic cell into a cell with no nucleus.

Ian Wilmut at the Roslin Institute in Scotland was assigned to a project in 1986. His goal was to create a sheep that produced a certain chemical in its milk. Than dolly was birth in 1987.

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Cloned Cat CC: The first cloned cat CC has quite a different character and fur colouring from its gene donor.

Cloned Mule : daho Gem”, first cloned animal in the horse family

Cloned pigs :- Some research focuses on pig clones as organ suppliers for human transplants.

Other cloned animals

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Why Pursue Animal Cloning Research

To generate groups of genetically identical animals for research purposes.

To rapidly propagate desirable animal stocks.

To improve the efficiency of generating and propagating transgenic livestock.

To produce targeted genetic alterations in domestic animals.

To pursue basic knowledge about cell differentiation.

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Natural cloning process

In nature, twins occur just after fertilization of an egg cell by a sperm cell. In rare cases, when the resulting fertilized egg, called a zygote, tries to divide into a two-celled embryo, the two cells separate.

Each cell continues dividing on its own, ultimately developing into a separate individual within the mother.

Since the two cells came from the same zygote, the resulting individuals are genetically identical

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Types of cloning

Recombinant DNA Technology or DNA Cloning.

Reproductive Cloning

Therapeutic Cloning

(Research cloning)

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What is DNA cloning?

DNA cloning is the isolation of a fragment or fragments of DNA from an organism and placing in a vector that replicates independently of chromosomal DNA.

The Recombinant DNA is propagated in a host organism, the resulting Clones are a set of genetically identical organisms which contain the recombinant DNA

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Three main purposes for cloning DNA

1) DNA sequencing Determining the sequence of the bases in the DNA can tell us about which proteins or RNAs are encoded and their sequences, which sequences control their expression (Gene promoters) and other control sequences), as well as any possible mutations which might alter their function.

Having access to the complete DNA sequence of an organism can help us decipher the biology of that organism.

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2) Protein production Isolating the gene which encodes a desired protein (hemoglobin, interferon) may allow that gene to be over-expressed so that the protein can be produced in bulk for study or use

3) Engineering animals/plants/proteins The ability to alter the properties of proteins as well as create genetically modified animals and plants (Transgenics) has lead to their use for research and for therapeutic and commercial purposes.

The technology may lead to the development of new therapies for the treatment of disease (GENE THERAPY).

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Advantages of cloning

Solution to infertility?

Provide organs for transplantation

Provide treatments for variety diseases

Better understanding of genetic diseases

Help improve lives

The healthiness of infants

Genetic modification / engineering

The healthiness of infants

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Disadvantages of cloning

Transgressing the nature

Cloning is “playing God”

Losing the diversity of genes

The great diseases and leading to extinction

Cause unbalance to the society

The uncertainty of science technology

Devastate parenting and family life

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Basic steps of cloning

Isolation of gene.

Insertion of isolated gene into vector

Transfer of recombinant vector into host cell

Identification & isolation of cells containing recombinant gene

Growth of host cells to amplify the cloned DNA number

Recovery of recombinant vector or expressions of target gene within host cell

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Step I isolation of target gene

Restriction endonuclease digestion

cDNA clones

Mechanical shearing

Chemical synthesis

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Restriction endonuclease digestion Two patterns:- 1) Blunt end pattern

-GGCC- -GG CC- - CCGG- -CC GG- (cleavage product)

2) Sticky or cohesive ends pattern -GAATTC- -GAATTC- - CTTAAG- -CTTAAG- (cleavage product)

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Step 2 Insertion of target fragment in to a vector

Direct ligation of cohesive termini

Blunt end ligation

Linker and adapters

Homopolymer tailing

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Step 3 Transformation

Target gene contaning plasmid (recombinant vector )

Uptake by E.Coli under pre treatement with calcium chloride.

Linear DNA is infective in transformation.

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Step 4 Identification & isolation of recombinant gene.

Colony hybridization (Nucleic acid hybridization) bye replica plate Method

Hybrid release translation (HRT method)

Immunochemical detection

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Step 5 expression of cloned gene (within the host)

Cloned gene is expressed in host by transcription & translation under machinery (operon system) of host.

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Future applications of cloning

Medical Purposes

Research for bio transplantation

Therapeutic cloning of stem cells from human embroys.

Clone for laboratory

Agricultural applications

Genetically modified cattle for specific purpose

Cloning the elite

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Ethics of Human cloning

The American Association for the Advancement of Science (AAAS) and other scientific organizations have made public statements suggesting that human reproductive cloning be banned until safety issues are resolved.

Gregory Stock is a scientist and outspoken critic against restrictions on cloning research. According to the US Food and Drug Administration, food coming from cloned animals is safe to eat.

Richard Nicholson of the British Bulletin of Medical Ethics said that cloning research may well be “sowing the seeds of our own destruction.”

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Benefits of human cloning

Rejuvenation

Heart attack Treatment

Human stem cells

Infertility treatment

Use in Surgeries

Defective genes

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Screening strategies

Screening by hybridization.

Immunological screening.

Screening by alternative ligands.

Screening by PCR.

Screening by functional complementation

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Flow chart of screening methods

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Screening by Hybridization

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Immunological screening

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Immunological screening for expressed genes.

A filter is taken from a Petridish containing the recombinants

(usually cDNA/λ constructs).

Protein and cell debrisadhere to the filter. Plaques expressing

the target protein (+) are indistinguishable from the others (–) at

this stage.

The filter is incubated with a primary antibody that is specific

for the target protein.

This is then complexed with radiolabelled protein A, and an

autoradiograph is prepared

Immunological screening

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Subcloning

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Definition:- Sub cloning is a basic procedure in molecular biology required to move inserts from one vector to another to gain the desired functionality to study your insert.

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Procedure of Subcloning

Requirements

Parent vector & destination vector.

Restriction endonuclease enzyme.

Purification- Gel electrophoresis

Amplification.

DNA ligase.

Phosphatase enzyme.

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Restriction enzymes are used to excise the gene of interest (the insert) from the parent.

A common purification method is gel isolation. The number of copies of the gene is then amplified using Polymerase Chain Reaction (PCR).

Simultaneously, the same restriction enzyme are used to digest (cut) the destination.

CIAP is also added to prevent self-ligation of the destination vector. The digested destination vector is isolated/purified and amplified using PCR. The two PCR products are mixed together with DNA ligase in ratio of 3:1. Amplification of product plasmid

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The plasmid is often transformed into a bacterium like E. coli. Ideally when the bacterium divides the plasmid should also be replicated. A marker gene is used in the destination vector for selection.

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3. Gel purifying DNA fragments:-

Use a low melt agarose gel

Don't waste your time by running the gel slowly.( Use about 5

volts per cm, i.e. 100 V for a minigel box, 150 V for a medium gel

box)

When the gel is done, place it on a clean piece of saran wrap on a

long wave (365 nm) UV box and take a picture.

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Preparation of DNA fragments for ligation

1. Restriction digests:

For double digests

2. Playing with the ends.

Blunting 5' overhangs

Blunting 3' overhangs

Blunting both a 3' and 5' end (T4 DNA polymerase)

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DNA cloning is to use DNA method to clone a gene to a vector,

screening and sequencing confirmation is usually required because

PCR may introduce mutations.

Subcloning is to use restriction enzymes to move one gene from

one vector to another vector. Screening and sequencing

confirmation may not be necessary.

Difference between cloning and subcloning

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Summary

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Chugh P. et al.2003. Cloning of gene, Indian Journal of Microbiology. 21(2):77-81.

Fromme T. et al. 2007. Rapid single steps subcloning , Journal of Biochemical Engineering,10(4): 1-7

Krishnan P.N.et al. 1984. Cloning American Association , 32,135-140

Kumives N. et al. 2007 ,Gene subcloning Chemical Enginering Education, 214-217

Micheal A.et al. 2002. Gene cloning , Biochem Journal , 382:131-135

References

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Meskiena R. et al. 2003 ,Cloning and Analysis ISSN: 1392-1396

Quinn L .et al. 2005 . Seamless gene cloning and gene fusion , ELESVIER Ltd. 23 : 199-208

Zhang D. 2005 . Rapid and efficient subcloning of DNA without dephosphorilation, Human Press Ltd. 29,111-118.

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