Gene Cloning

Gene Cloning – Enzymatic cleavage of DNA, Restriction enzymes (Endonucleases) and Ligation.

Gene Cloning

To clone means to make identical copies. Gene cloning means production of a number of similar copies of a required gene. DNA cloning involves separating a specific gene or DNA segment from a larger chromosome, attaching it to a small carrier DNA. The resultant hybrid DNA is called recombinant DNA, which is transferred to a proper host (bacteria, virus or yeast) and replicated to make multiple copies of the selected gene.

This technology has made it possible to isolate, clone and produce DNA for all the genes in appropriate quantity so that they can be sequenced and characterized. Similarly, some of the genes which are expressed at very low level, can be cloned and desired amount of recombinant proteins can be produced.

Gene cloning involves the following steps

1. Cutting the DNA to be cloned from the chromosomal using sequence specific Restriction Endonuclease.

2. Selecting a cloning vector (a small molecule capable of self-replicating inside host cells), and cutting the cloning vector with the same restriction endonuclease to produce sticky ends.

3. Incubating the vector and subject DNA to join together DNA ligase. The resultant DNA is called recombinant DNA.

4. Transferring the reconbinant DNA to an appropriate host such as bacteria, virus or yeast which will provide necessory biomachinary for DNA replication.

5. Identifying the host cells that contain the recombinant DNA.

Enzymatic Cleavage of DNA
To cut the DNA at specific sites Restriction endonucleases are used. (RENs).
Restrictions Endonucleases:
A Restriction Endonuclease is an enzyme that cuts DNA at specific recognition points known as Restriction sites. These are most important groups of enzymes for manipulation of DNA. These enzymes were discovered in Escherichia coli. In bacteriophages these enzymes restrict the replication of viral DNA. Many types of restriction endonucleases were isolated. RENs were named based on the bacterial from which they were isolated.
The first letter of the enzyme indicates the genus name and next two letters the species name, followed by strain name and finally a number indicating the order of discovery. Eg. EcoRI: here E represents Escherichia, co represents coli, R represents the strain and I represents the first endonuclease.

Types of Endonucleases: over 3000 RENs have been studied in detail and more than 600 are available commercially. Naturally occurring Endonucleases are categorized in to four groups namely type I, II, III and IV based on their nature of their restriction sites. All types of enzymes recognize a specific DNA sequence and cleave that DNA at that point. They differ in their recognition sequence and cofactor requirements.

Type I RENs cuts DNA at random location as far as 1000 or more Basepairs from the recognition site. Type II cuts approximately 25 base pairs from the site. Type I and Type III require ATP. They are large enzymes with multiple subunits.
Type II RENs cut the DNA with in the recognized sequence without the need of ATP. They are smaller and simpler. Hence they are predominantly used in biotechnology.
Most of the Type II RENs generate Sticky ends. The open ends of the DNA molecule after the cut are called as Sticky ends.
The Length of restriction recognition sites varies. The Enzyme EcoRI, SacI etc recognize 6 basepair sequence of DNA. Most of the recognition sequences are palindromes – they read the same forward and backward.

Some of the important RENS and their restriction sites.
Enzyme Source Recognition Sequence Cut
EcoRI
Escherichia coli
5'GAATTC
3'CTTAAG 5'---G AATTC---3'
3'---CTTAA G---5'
EcoRII
Escherichia coli
5'CCWGG
3'GGWCC 5'--- CCWGG---3'
3'---GGWCC ---5'
BamHI
Bacillus amyloliquefaciens
5'GGATCC
3'CCTAGG 5'---G GATCC---3'
3'---CCTAG G---5'
HindIII
Haemophilus influenzae
5'AAGCTT
3'TTCGAA 5'---A AGCTT---3'
3'---TTCGA A---5'
TaqI
Thermus aquaticus
5'TCGA
3'AGCT 5'---T CGA---3'
3'---AGC T---5'


For convenience it is usual practice to simplifly the description of recognition sequences by showing only one strand of DNA, which runs in the 5’ to 3’ direction. Thus the EcoRI recognition sequence would be shown as G\AATTC.
Restriction enzymes with same sequence specificity and cut site are known as isochizomers. Enzymes that recognize the same sequences but cleave at different points are known as Neochizomers. Under extreme conditions like rise in pH, Low ionic strength) RENs are capable of cleaving sequences which are similar but not identical to their definied recognition sequences.

Applications of Restriction Endonucleases:
1. RENs formed in different bacteria can be used to break the DNA of any organism and the required DNA segment can be introduced in the DNA of another organism to produce Recombinant DNA
2. Restriction enzymes are highly useful to get desirable DNA segments. This is because these enzymes break the DNA at specific sites.
3. The DNAsegment isolated with the help of restriction enzymes can be used in DNA probe, genomic libraries and cDNA libraries.
4. With the isolated DNA segments, mRNA can be transcribed with which required proteins can be synthesized.
5. In restriction Fragment Length Polymorphism (RFLP) also restriction enzymes are very useful.

Enzymatic ligation of DNA
To hybridize (attach) the DNA fragments formed by the RENs to a plasmid DNA, the ends of both DNA are to be attached by forming hydrogen bonds and diester bonds. This process of attachment of the DNA fragments is known as Ligation. It is done by certain specialized enzymes called Ligases.

Ligase: (ligare = to glue together) is a special type of enzyme that can link together two DNA strands that have double strand break.
Mertz and Davis, 1972 first succeeded in producing rDNA in Escherichia coli by ligation of the sticky ends of DNA with ligase.
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3’hydroxyl ends of one nucleotide with the 5’ phosphate end of another. ATP is required for ligation. Ligase will also work with blunt ends, but neets high concentrations of enzymes.
In mammals there are four specific types of ligases.ie. DNA ligases, I, II, III and IV. LIgases can be classified into two groups on their requirement for ATP and NAD+ as co factors. All eukaryotic enzymes are ATP dependent, whereas most prokaryotic enzymes require NAD+ for their activity.
Most experiments use T4 DNA ligase (isolated from bacteriophase T4) which is most active at 25oC. High temperatures disrupts hydrogen bonding. The commonly available DNA ligases were originally discovered in bacteriophase T4, E.coli and other bacteria.

Applications of Ligases
DNA ligases are essential tools in modern molecular biology for generating rDNA sequences such as
1. Joining double stranded DNA with cohesive or blunt ends
2. Joining of oligonucleotide linkers or adaptors to blunt ended DNA
3. Repairing in duplex DNA, RNA or DNA-RNA hybrids
4. DNA ligases are used with restriction enzyes to insert DNA fragments often genes into plasmids.

Transgenesis and Production of transgenic animals (Fish and Goat).

Transgenesis is the process of introducing an exogenous gene – called transgene- into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. A transgene is the name given to the introduced DNA. The term transgenesis was coined by Gordon and Ruddle in 1981.

Animals produced through transgenesis are called Transgenic animals. Transgenic animals are genetically modified organisms with a new hereditary character.
Transgenic animals can be used to produce valuable products. Many proteins produced by transgenic animals are important for medical applications. For example, a transgenic pig has been produced with the ability to synthesize human hemoglobin for use as a blood substitute. Transgenic goat has been developed to produce a protein needed by the patients suffering from cystic fibrosis. Transgenisis is essential for improving the quality and quantity of the eggs, meat, milk etc, in addition to drug resistant animals.
The Mouse is the first animal used for transgenesis. RD Palmiter and RL Brinter (1982) isolated gene for growth hormone in human being. This gene was ligated with plasmid pBR322 to produce rDNA. It was transferred to the zygote of a mouse invitro. The embryo was implanted in the uterus of a foster mouse. Then the new born mouse was found to be transgenic which contained a gene from humans.

Methods of creation of transgenic Animals
There are three methods used for creations of transgenic animals are DNA micro injection, Embryonic stemcell-mediated gene transfer and Retrovirus mediated gene transfer.

DNA micro injection
Introduction of transgene by microinjection involves the following procedure. A young female mouse is given the FSH (follicle stimulating hormone) and HCG (human chorionic gonadotropin). Thus the mouse produces 30-35 ova. It was allowed to mate with a male. Then the fertilized ova are collected from the fallopian tubes. The transgene is introduced into the male pronucleus by using
micro injection needle. After amphimixis the embryo is allowed to devide. Then the embryo was implanted into the uterus of a foster mother. The new borns are called transgenic mice.

Hence DNA micro injection method is a random method. Success rate is very poor. This method has many disadvantages. The introduced DNA may not insert into the genome of the host. The foster mother may not accept the introduced fertilized ovum for further development. The introduced DNA may not express the desired trait. . A major advantage of this method is its applicability to a wide variety of species..

Embryonic stemcell-mediated gene transfer
The Recombinant DNA is transferred into embryo stem cells (ES). The cells are then cultured in the laboratory and those expressing the desired protein are selected. These modified ES cells are incorporated into the cavity of the embryo. This embryo is raised in a foster mother. The resulting transgenic animal will be a mosaic, because only a small proportion of the cells in its body will be expressing the protein.
Through this method transgenic goats and cows can now be designed to produce human proteins like blood clotting factors intheir milk.

Retroviral vector method:
Small fragments of DNA (8kb) can be effectively transferred by retrovirus. This method is not suitable for the transfer of large fragments of DNA. The main drawback in this method is the risk of retroviral contamination in products (such as human food) produced by transgenic animals. Hence this method is not popular in transgenesis.