introduction recombinant dna technology has revolutionized biochemistry since it came into being in...

IntroductionIntroduction

Recombinant DNA technology has revolutionized biochemistry since it came into being in the 1970s.

The genetic endowment of organisms can now be precisely changed in designed ways.

Recombinant DNA technology is a fruit of several decades of basic research on DNA, RNA, and viruses

The pioneering work of Paul Berg, Herbert Boyer, and Stanley Cohen in the 1970s led to the development of recombinant DNA technology.

It has permitted biology to move from an exclusively analytical science to a synthetic one.

A recombinant DNA molecule is produced by joiningtogether two or more DNA segments usually originating fromdifferent organisms.

More specifically, a recombinant DNA molecule is a vector into which the desired DNA fragment has been insertedto enable its cloning in an appropriate host.

This is achieved by using specific enzymes for cutting the DNA (restriction enzymes) into suitable fragments and then for joining together the appropriate fragments (ligation).Thus it is based on Dna enzymology.

Recombinant DNA molecules are produced with one of the following three objectives

(1) To obtain a large number of copies of specificDNA fragments,

(2) To recover large quantities of the protein produced by the concerned gene, or

(3) To integrate the gene in question into the chromosome of a target organism where it expresses itself.

The Recombinant DNA technology is useful

– Identifying genes– Isolating genes– Modifying genes– Re-expressing genes in other hosts or

organisms

Cloning Cloning

The DNA segments are integrated into a self replicating DNA molecule called vector; most commonly used vectors are either bacterial plasmids or DNA viruses.

All these steps concerned with piecing together DNA segments of diverse origin and placing them into a suitable vector together constitute recombinant DNA technology

The DNA segment to be cloned is called DNA insert.

Recombinant DNAs are introduced into a suitable organism, usually a bacterium.

This organism is called host, while the process is called transformation.

The transformed host cells are selected and cloned.

Often DNA or gene cloning is taken to include both the development of recombinant DNAs as well as their cloning in a suitable host.

Similarly, often the term recombinant DNA technology is used as a synonym for DNA or gene cloning used in the broader sense.

A rather popular term for these activities is genetic engineering.

CLONE CLONE

A clone consists of asexual progeny of a single individual or cell, while the process/technique of producing a clone is called cloning.

As a result, all the individuals of a clone have the same genotype, which is also identical with that of the individual from which the clone was derived.

Therefore gene or DNA cloning produces large numbers of copies of the gene/ DNA being cloned.

Recombinant dna technologyRecombinant dna technology

Recombinant DNA technology, often known as Gene cloning or cloning which involves - isolation of a fragment of a genome (an entire gene, or other sequence of interest), and incorporation in a "replicon or vector", that is replicated independently of the original DNA molecule.

Procedure Procedure

It includes following steps: 1. Isolation of plasmid DNA (or any vector) 2. Digestion with restriction enzymes 3. Purification of the required fragment 4. Ligation of fragment into new vector 5. Transformation of E.coli strain with new plasmid 6. Selection of recombinant plasmids 7. Analysis of recombinant plasmids 8. Replication of recombinant strains 9. Production of identical copies

Tools for Gene Cloning or DNA recombinant technology:

1. Vector 2. Host Organisms 3. Restriction enzymes 4. Selection of markers for cloned colony

screening

1. Vectors:A plasmid or a bactriophage into which foreign DNA can be introduced for the purpose of cloning.

In every case, the recombinant DNA must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporating the DNA in a Vector.

The most common vectors used are Plasmids Cosmids λ phage DNA Retro viruses & Yeast artificial chromosome.

(a) Plasmids: Plasmids are molecules of DNA that are found in

bacteria but are separate from the bacterial chromosome. They are circular DNA molecule, a few thousand base pairs and usually carry only one or a few genes. DNA fragment of upto 5Kb can be inserted into a plasmid.

The properties of Plasmids which make them ideal for cloning includes:

1. Origin of DNA replication -

That helps in replication of the DNA fragment once inserted into the plamid

2. A dominant selectable marker -

Usually resistant to an antibiotic Ampicilin - R or Tetracycline-R) that helps in detecting the presence or absence of recombinant DNA in the host organisms.

3. . Unique restriction cleavage sites: -

This helps in the digestion of the plasmid and DNA of interest from the same site, so that they can be ligated together. Moreover, these restriction sites sometimes present in a gene like Lac-Z gene, whose disruption on digestion acts as scorable marker for confirmation of recombination.

(b) Cosmids: A genetically engineered plasmid that contains the COS sites of

ephage DNA, a drug-resistance gene. c) Phage A DNA: Phage A DNA is Bacteriophage DNA, which has copy

numbers, an Ampicillin-resistance gene and multiple cloning site or poly-linker .

(d) Retroviruses: Retroviruses can infect virtually any type of mammalian cell

and is a common vector to clone DNA in mammalian cells. Contains reverse transcriptase.

Enzymes required:

A battery of enzymes are required to carry the process.

Restriction Endonucleases: These are called DNA scissors, which cut DNA at specific sequences, called

restriction sites, within the sequences. They are totally different from DNases, which do not recognize specific sites and randomly cleave DNA

into variable series of small fragments. 2. DNA ligase These enzymes are responsible for ligating the vector and DNA fragment already digested with the same restriction enzyme. DNA restriction

Endonuclease and DNA ligases works in tandem to form the recombinant DNA.

3. Alkaline phosphatase Removes Phosphate groups at the 3' end of the fragments,

which helps in ligation of the two fragments by adapters. 4. Polynucleotide kinase Adds Phosphate groups at the 3' end of the fragments, which

helps in the ligation of the two fragments by Poly linkers. 5. Terminal transferase Add Poly C at the 3' end of vector and Poly G at 5' end of

the fragments, so that they can be ligated without the help of linkers or adapters.

6. Reverse transcriptase This particular enzyme carry the c-DNA synthesis from

the m-RNA.This helps in cloning of mature transcript sequence in the form of cDNA, as prokaryotic host i.e. bacteria can't process eukariotic gene II cloned as DNA.

7. Taq polymerase Used for PCR amplification of the DNA fragment which is

then eluted from the gel and is used in cloning. 8. RNAse A/RNAse H

It removes RNA from the sample mixture.

RESTRICTION ENZYMES Certain endonucleases—enzymes that cut DNA at specific DNA

sequences within the molecule—are a key tool in recombinant DNA research. These enzymes were called restriction enzymes because their presence in a given bacterium restricted the growth of certain bacterial viruses called bacteriophages.

Restriction endonucleases recognize short stretches of DNA (generally 4/6 bp) that contain specific nucleotide sequences. These sequences, which differ for each restriction endonuclease, are palindromes, that is, they exhibit twofold rotational symmetry . This means that, within a short region of the double helix, the nucleotide sequence on the “top” strand, read 5′→3′, is identical to that of the “bottom” strand, also read in the 5′→3′ direction.

Restriction enzymes are named after the bacterium from which they are isolated.

EcoRI is from Escherichia coli, and BamHI is from Bacillus amyloliquefaciens. The first three letters in the restriction enzyme name consist of the first letter of the genus (E ) and the first two letters of the species (co ). These may be followed by a strain designation (R ) and a roman numeral (I ) to indicate the order of discovery (eg, EcoRI, EcoRII ). Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is typically 4–7 bp long. These DNA cuts result in blunt ends (eg, HpaI ) or overlapping (sticky or cohesive) ends (eg, BamHI). Sticky ends are useful in constructing chimeric DNA molecules

Competent cells:

They are the host organism cells in which recombinant DNA is transformed and replicated. These cells are highly capable of accepting DNA inserts, soaking in CaCl2, which makes their cell membranes permeable to DNA inserts.

Technique of DNA recombination:

Preparation of specific human gene:

Isolation of specific gene from human DNA is a very laborious process.this is like searching for a needle in haystack.this problem is solved by preparation of complimentary (c DNA)

Human DNA- or cDNA copied from mRNA using reverse transcriptase from retroviruses is used .

Preparation of chimeric DNA: Chimera in Greek =lions head ,goats body,serpent

tail.The vector carrying a foreign DNA is called chimeric DNA/hybrid dna.

A circular plasmid vector DNA is cut with restriction endonuclease(RE)if ECORI is used ,sticky ends are produced with TTAA sequence on one strand and AATT sequence on other strand.

The human DNA is also treated with RE.

Then the vector DNA & human cut piece DNA are incubated at 370 c together so that annealing takes place.The sticky ends of both vector & human DNA have complimentary sequences.

Then the DNA ligase is added which introduces phospho - diester linkages b/w vector & insert molecules .Thus the chimeric DNA is produced.

Cloning of chimeric DNA:A clone is a large population of identical

bacteria that commonly arise from a common ancester molecule .

Cloning allows the production of large number of identical DNA.

The hybrid molecules are amplified by the cloning techniques.

Transfection of vector into the host : The process by which plasmid is introduced into the

host is called transfection. Host E.coli cells and plasma vectors are incubated in

hypertonic medium containing calcium for a few minutes.

Then the calcium channels are opened ,plasmids are imbibed into the host cell.

The host cells are grown on agar plates containing growth medium containing desired colonies.

Plasmids carry Antibiotic Resistant Genes: Plasmid pBR- 325 vector contains

ampicillin,tetracycline,chloramphenicol resistance genes.

The RE Eco R1 will cleave the plasmid in the middle of cmr gene. When the foreign DNA is inserted ,the resistance against cmr is lost.

This insertional inactivation is marker for hybrid DNA

SELECTION OF COLONY HAVING DESIRED GENES :

The bacteria are now cultured in a medium contaning ampicillin and tetracycline that kill the wild bacteria.

Only the bacteria contaning plasmids grow. There will be many colonies where the vector does not

carry foreign DNA.these colonies are replicated onto another agar plate contaning cmr.

Since this insertion abolishes cmr resistance,the desired colonies are killed.

The colonies in the original plate correspond to dead colonies in the replica plate selected.They carry the foreign gene.

The selected colonies are further cultured to produce colonies.

Expression vectors : To produce the human proteins ,E coli carrying

the vector with the insert allowed to grow ,without any protein inhibitors .

Such a vector carrying the foreign gene which is translated into a protein is called expression vector.

The human proteins can be harvested from the bacterial culture.

Recombinant DNA technology has revolutionized the analysis of the molecular basis of life.

Complex chromosomes are being rapidly mapped and dissected into units that can be manipulated and deciphered.

The amplification of genes by cloning has provided abundant quantities of DNA for sequencing.

The Use of Recombinant DNA to Produce The Use of Recombinant DNA to Produce Human InsulinHuman Insulin

Protein hormone produced by beta cells of islets of Langerhans in the pancreas.

Regulates blood sugar by allowing uptake of glucose from bloodstream into body cells.

Patients with diabetes have insufficient or impaired production of insulin

Structure of InsulinStructure of Insulin

Two polypeptide chains; one with 21 amino acids and the second with 30 amino acids

Chains are linked via a disulfide bond

Gene encoding the insulin protein is found on chromosome 11

RECOMBINANT INSULINRECOMBINANT INSULIN

Restriction enzymes used to cut out insulin gene and to cut a bacterial (E. coli) plasmid at the same “sticky ends”.

Mutant strains of E. coli used to avoid bacteria attacking “foreign” genes

Insert insulin gene next to E. coli .

B-galactosidase gene which controls transcription Bacterial cells replicate and make copies of insulin gene

Restriction Fragment Length Restriction Fragment Length PolymorphismPolymorphism

A restriction fragment length polymorphism (RFLP) is a genetic variant that can be examined by cleaving the DNA into fragments (restriction fragments) with a restriction enzyme and seperated by gel electrophoresis.

The length of the restriction fragments is altered if the genetic variant alters the DNA so as to create or abolish a site of restriction endonuclease cleavage (a restriction site).

RFLP can be used to detect human genetic variations, for example, in prospective parents or in fetal tissue.

DNA variations resulting in RFLPDNA variations resulting in RFLP

Single base changes in DNA: About 90% of human genome variation comes in the form of single-nucleotide polymorphisms, that is, variations that involve just one base.

The alteration of one or more nucleotides at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site can also be created by the same mechanism. In either case, cleavage with an endonuclease results in fragments of lengths differing from the normal, which can be detected by DNA hybridization .

The altered site can be mutation causing or distant from mutation site.

Tandem repeats These are short sequences of DNA at scattered locations in the

genome, repeated in tandem (one after another). The number of these repeat units varies from person to person, but is unique for any given individual and, therefore, serves as a molecular fingerprint.

Cleavage by restriction enzymes yields fragments that vary in length depending on how many repeated segments are contained in the fragment. Variation in the number of tandem repeats can lead to polymorphisms (Figure 33.14). Many different VNTR loci have been identified, and are extremely useful for DNA fingerprint analysis

New vistas in r DNA TECHNOLOGYNew vistas in r DNA TECHNOLOGY

Large amounts of protein can be obtained by expressing cloned genes or cDNAs in bacteria or eukaryotic cells.

Hormones, such as insulin, and antiviral agents, such as interferon, are being produced by bacteria.

Tissue plasminogen activator, which is administered to a patient after a heart attack, is made in large quantities in mammalian cells.

A new pharmacology, using proteins produced by recombinant DNA technology as drugs, is beginning to significantly alter the practice of medicine.

Recombinant DNA technology is also providing highly specific diagnostic reagents, such as DNA probes for the detection of genetic diseases, infections, and cancers.

Human gene therapy has been successfully initiated.

White blood cells deficient in adenosine deaminase, an essential enzyme, are taken from patients and returned after being transformed

in vitro to correct the genetic error.

Agriculture, too, is benefiting from genetic engineering. Transgenic crops with increased resistance to insects, herbicides, and drought have been produced.

We can construct new genes with designed properties by making three kinds of DNA directed changes: deletions, insertions,and

substitutions. Gene mapping recombinant technology is used to locate genes on

chromosomes New insights are emerging, as exemplified by the discovery of introns

in eukaryotic genes. Novel Proteins Can Be Engineered by Site-Specific Mutagenesis

It is used for development of DNA vaccines and biosensors.

CONCLUSIONCONCLUSION

Recombinant DNA technology is not only an important tool in scientific research, but has also resulted in enormous progress in the diagnosis and treatment of certain diseases and genetic disorders in many areas of medicine.

Thus Recombinant DNA technology is beginning to significantly alter the practice of medicine by providing new diagnostic and therapeutic agents and revealing molecular mechanisms of disease.