Recombinant DNA Technology and its Applications - Tools for Recombinant DNA Technology

Recombinant DNA Technology (With Diagram)

Recombinant DNA Technology (With Diagram)

 

Tools for Recombinant DNA Technology:

1WDNA technology utilizes a number of biological tools to achieve its objectives, most important of them being the enzymes.

Important biological tools for rec DNA technology are:

(A) Enzymes:

a. Restriction Endonucleases

b. Exonucleases

c. DNA ligases

d. DNA polymerase

 

(B) Cloning Vector

(C) Host organism

(D) DNA insert or foreign DNA

(E) Linker and adaptor sequences.

 

An account of all these biological tools of genetic engineering is given below:

(A) ENZYMES:

A number of specific enzymes are utilized to achieve the objectives of rec DNA technology.

 

 utilize 활용하다

 

The enzymology of genetic engineering includes the following types of enzymes:

(a) Restriction Endonuclease:

These enzymes serve as important tools to cut DNA molecules at specific sites, which is the basic need for rec DNA technology.

 

These are the enzymes that produce internal cuts (cleavage) in the strands of DNA, only within or near some specific sites called recognition sites/recognition sequences/ restriction sites target sites. Such recognition sequences are specific for each restriction enzyme.

 

Restriction endonuclease enzymes are the first necessity for rec DNA technology.

 

The presence of restriction enzymes was first of all reported by W. Arber in the year 1962. He found that when the DNA of a phage was introduced into a host bacterium, it was fragmented into small pieces. This led him to postulate the presence of restriction enzymes. The first true restriction endonuclease was isolated in 1970s from the bacterium E. coli by Meselson and Yuan. Another important breakthrough was the discovery of restriction enzyme Hind-II in 1970s by Kelly, Smith and Nathans. They isolated it from -the bacterium Haemophilus influenza. In the year 1978, the Nobel Prize for Physiology and Medicine was given to Smith, Arber and Nathans for the discovery of endonucleases.

 

Types of Restriction Endonucleases:

There are 3 main categories of restriction endonuclease enzymes:

Type-I Restriction Endonucleases

Type-II Restriction Endonucleases

Type-III Restriction Endonucleases

 

Type-I Restriction Endonucleases:

These are the complex type of endonucleases which cleave only one strand of DNA. These enzymes have the recognition sequences of about 15 bp length (Table 1).

They require Mg++ ions and ATP for their functioning. Such types of restriction endonucleases cleave the DNA about 1000 bp away from the 5′ end of the sequence ‘TCA’ located within the recognition site. Important examples of Type-I restriction endonuclease enzyme are EcoK, EcoB, etc.

 

 cleave 쪼개다

 

 

Type-II Restriction Endonucleases:

These are most important endonucleases for gene cloning and hence for rec DNA technology. These enzymes are most stable. They show cleavage only at specific sites and therefore they produce the DNA fragments of a defined length. These enzymes show cleavage in both the strands of DNA, immediately outs.de then- recognition sequences. They require Mg++ ions for their functioning.

 

cleavage 분열

fragment 조각

 

Such enzymes are advantageous because they don’t require ATP for cleavage and they cause cleavage in both strands of DNA. Only Type II Restriction Endonucleases are used tor gene cloning due to their suitability.

 

The recognition sequences for Type-II Restriction Endonuclease enzymes are in the form of palindromic sequences with rotational symmetry, i.e., the base sequence .n the first half of one strand of DNA is the mirror image of the second half of other strand of that DNA double helix (Fig. 2). Important examples of Type-II Restriction endonucleases include Hinfl, EcoRI, PvuII, Alul, Haelll etc.

 

Type-III Restriction Endonucleases:

These are not used for gene cloning. They are the intermediate enzymes between Type-I and Type-II restriction endonuclease. They require Mg++ ions and ATP for cleavage and they cleave the DNA at well-defined sites in the immediate vicinity of recognition sequences, e.g. Hinf III, etc.

 

 

Nature of cleavage by Restriction Endonucleases:

The nature of cleavage produced by a restriction endonuclease is of considerable importance.

 

They cut the DNA molecule in two ways:

i. Many restriction endonucleases cleave both strands of DNA simply at the same point within the recognition sequence. As a result of this type of cleavage, the DNA fragments with blunt ends are generated. PvuII, Haelll, Alul are the examples of restriction endonucleases producing blunt ends. Blunt ends may also be referred to as flush ends.

 

 

ii. In the other style of cleavage by the restriction endonucleases, the two strands of DNA are cut at two different points. Such cuts are termed as staggered cuts and this results into the generation of protruding ends i.e., one strand of the double helix extends a few bases beyond the other strand. Such ends are, called cohesive or sticky ends.

 

 

Such ends have the property to pair readily with each other when pairing conditions are provided. Another feature of the restriction endonucleases producing such sticky ends is that two or more of such enzymes with different recognition sequences may generate the same sticky ends.

 

(b) Exonucleases:

Exonuclease is an enzyme that removes nucleotides from the ends of a nucleic acid molecule. An exonuclease removes nucleotide from the 5′ or 3′ end of a DNA molecule. An exonuclease never produces internal cuts in DNA.

 

In rec DNA technology, various types of exonucleases are employed like Exonuclease Bal31, E. coli exonuclease III, Lambda exonuclease, etc. Exonculease Bal31 are employed for making the DNA fragment with blunt ends shorter from both its ends. E coli Exonuclease III is utilized for 3’end modifications because it has the capability to remove nucleotides from 3′-OH end of DNA. Lambda exonuclease is used to modify 5′ ends of DNA as it removes the nucleotides from 5′ terminus of a linear DNA molecule.

 

(c) DNA ligase:

The function of these enzymes is to join two fragments of DNA by synthesizing the phosphodiester bond. They function to repair the single stranded nicks in DNA double helix and in rec DNA technology they are employed for sealing the nicks between adjacent nucleotides. This enzyme is also termed as molecular glue.

 

(d) DNA polymerases:

These are the enzymes which synthesize a new complementary DNA strand of an existing DNA or RNA template. A few important types of DNA polymerases are used routinely in genetic engineering.

 

synthesize 합성하다

 

 

One such enzyme is DNA polymerase ! which , prepared from E coli. The Klenow fragment of DNA polymerase-I .s employed to make the protruding ends double-stranded by extension of the shorter strand. Another type of DNA polymerase used in genetic engineering is Taq DNA polymerase which is used in PCR (Polymerase Chain Reaction). Reverse transcriptase is also an important type of DNA polymerase enzyme for genetic engineering. It uses RNA as a template for synthesizing a new DNA strand called as cDNA a complementary DNA). Its main use is in the formation of cDNA libraries. Apart from all these above mentioned enzymes, a few other enzymes also mark their importance in genetic engineering.

 

Reverse transcriptase 역전사효소

 

A brief description of these is given below:

(a) Terminal deoxynucleotidyl transferase enzyme:

It adds single stranded sequences to 3′-terminus of the DNA molecule. One or more deoxynbonucleotides (dATP, dGTP, dl IP, dCTP) are added onto the 3′-end of the blunt-ended fragments.

(b) Alkaline Phosphatase Enzyme:

It functions to remove the phosphate group from the 5′-end of a DNA molecule.

(c) Polynucleotide Kinase Enzyme:

It has an effect reverse to that of Alkaline Phosphatase, i.e. it functions to add phosphate group to the 5′-terminus of a DNA molecule

(B) Cloning Vectors:

It is another important natural tool which geneticists use in rec DNA technology. The cloning vector is the DNA molecule capable of replication in a host organism, into which the target DNA is introduced producing the rec DNA molecule. 

A cloning vector may also be termed as a cloning vehicle or earner DNA or simply as a vector or a vehicle a great variety of cloning vectors are present for use with E. coli is the host organism.

 

However under certain circumstances it becomes desirable to use different host for cloning experiments. So, various cloning vectors have been developed based on other bacteria like Bacillus, Pseudomonas, Agrobacterium, etc. and on different eukaryotic organisms like yeast and other fungi.

The cloning vector which has only a single site for cutting by a particular restriction endormclease is Considered as a good cloning vector. Different types of DNA molecules may be used as cloning vehicles such as they may be plasmids, bacteriophages, cosmids, phasmids or artificial chromosomes.

(C) Host Organism:

A good host organism is an essential tool tor genetic engineering. Most widely used host for rec DNA technology is the bacterium E. coli. because cloning and isolation of DNA inserts is very easy in this host. A good host organism is the one winch easy to transform and in which the replication of rec DNA is easier. There should not be any interfering element against the replication of rec DNA in the host cells

(D) DNA Insert Or Foreign DNA:

The desired DNA segment which is to be cloned is called as DNA insert or foreign DNA or target DNA. The selection of a suitable target DNA is the very first step of rec DNA technology. The target DNA (gene) may be of viral, plant, animal or bacterial origin.

 

Following points must be kept in mind while selecting the foreign DNA:

1. CD It can be easily extracted from source.

2. It can be easily introduced into the vector.

3. The genes should be beneficial for commercial or research point of view.

A number of foreign genes are being cloned for benefit of human beings. Some of these DNA inserts are the genes responsible for the production of insulin, interferon’s, lymphotoxins various growth factors, interleukins, etc.

 

(E) Linker and Adaptor Sequences:

Linkers and adaptors are the DNA molecules which help in the modifications of cut ends of DNA fragments. These can be joined to the cut ends and hence produce modifications as desired.

 

modification 수정

 

Both are short, chemically synthesized, double stranded DNA sequences. Linkers have (within them) one or more restriction endonuclease sites and adaptors have one or both sticky ends. Different types of linkers and adaptors are used for different purposes.

 

 

Linkers contain target sites for the action of one or more restriction enzymes. They can be ligated to the blunt ends of foreign DNA or vector DNA (Fig. 5a). Then they undergo a treatment with a specific restriction endonuclease to produce cohesive ends of DNA fragments EcoRI-linker is a common example of frequently used linkers.

 

undergo 겪다

 

 

 

 

Adaptors are the chemically Synthesized molecules which have pre-formed cohesive ends (Fig. 5b). Adaptors are employed for end modification in cases where the recognition site for restriction endonuclease enzyme is present within the foreign DNA.

The foreign DNA is ligated with adaptor on both ends. This new molecule, so formed, is then phosphorylated at the 5′-terminii. Finally foreign DNA modified with adaptors is integrated into the vector DNA to form the recombinant DNA molecule.