Biochemistry 26 Recombinant DNA Technology

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Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

Description of Module

Subject Name Biochemistry

Dr. Vijaya Khader Dr. MC Varadaraj Paper Name 12 Biochemical Techniques

Module Name/Title 26 Recombinant DNA Technology

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

1. Objectives

 Understanding the elements and tools of recombinant technology  Various steps in Recombinant DNA technology  Expression of prokaryotic gene

2. Concept Map

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

3. Description

3.1 Genetic engineering and Recombinant DNA technology

The manipulation of genes isolated from an organism to alter some specific characteristics attributed by the gene is what is generally referred to as genetic engineering. Recombinant DNA technology is a component of genetic engineering. It refers to the process of cutting a known or a target DNA sequence from one organism and introducing it further into another organism thereby altering the genotype of the recipient organism. Genetic engineering finds tremendous applications in fields of medicine, agriculture, and industry.

3.2 What is a Recombinant DNA

Recombinant DNA or rec DNA, is a form of artificial DNA that is made through the combination of one or more DNA strands or insertion of one portion of a strand into another, therefore leading to combination of DNA sequences within different species.

3.3 Elements of recombinant DNA technology

Listed below are some of the key elements required for recombinant technology:

(A) Enzymes:

a. Restriction Endonucleases

b. Exonucleases

c. DNA ligases

d. DNA polymerase

e. Other enzymes

(B) Cloning Vector

(D) DNA insert or foreign DNA

(E) Linker and adaptor sequences.

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

3.3.1 Enzymes

A number of enzymes are used in recombinant DNA technology. They are briefly described below:

(a) Restriction Endonuclease:

These enzymes cut DNA molecules at specific sites. The cuts (cleavage) in DNA strands are made only within or near some specific sites called recognition sites/ recognition sequences/ restriction sites. Recognition sites are specific for each restriction enzyme. There are 3 main categories of restriction endonuclease enzymes, namely Type-I, Type-II and Type-III Restriction Endonucleases (Table 1).

Table 1 Different types of restriction endonucleases

Type-I Restriction Type-II Restriction Type-III Restriction Endonucleases Endonucleases Endonucleases

Properties  These endonucleases  These enzymes are most  These are not used for cleave only one strand stable. gene cloning. of DNA.  They show cleavage only  They are the  These enzymes have at specific sites intermediate enzymes the recognition  They produce the DNA between Type-I and sequences of about 15 fragments of a defined Type-II restriction bp length length. endonuclease.  These enzymes show cleavage in both the

strands of DNA,

immediately outside then- recognition sequences.  Only Type II Restriction Endonucleases are used for gene cloning due to their suitability. Cleavage pattern  DNA cleavage takes  The recognition  They cleave the DNA at place about 1000 bp sequences for Type-II well-defined sites in the away from the 5′ end of Restriction Endonuclease immediate vicinity of the sequence ‘TCA’, enzymes are in the form recognition sequences,

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

which is located within of palindromic sequences e.g. Hinf III, etc. the recognition site with rotational symmetry Requirement of Mg  They require Mg2+ ions  They require Mg2+ ions  They require Mg2+ ions 2+ for their functioning for their functioning for cleavage

Requirement of ATP  They require ATP for  They don’t require ATP  They require ATP for their functioning for cleavage cleavage Examples  EcoK, EcoB  Hinfl, EcoRI, PvuII, Alul, Haelll

Nature of cleavage by Restriction Endonucleases:

Restriction endonuclease can cut the DNA molecule in two ways:

1. They can cleave the two DNA strands at two different points. Such cuts are termed as staggered cuts and this results into the generation of protruding ends or sticky ends. Such ends can pair readily with each other upon being provided with suitable pairing conditions. 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.

2. They can cleave both strands of DNA simply at the same point within the recognition sequence. Thus, DNA fragments with blunt ends are generated. PvuII, Haelll, Alul are the examples of restriction endonucleases producing blunt ends.

Figure 1 below shows the nature of cleavage by restriction enzymes to produce blunt and sticky ends.

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

Figure 1 Cleavage by restriction endonucleases: sticky and blunt ends

(b) Exonucleases: Exonuclease, as the name suggests, is an enzyme that removes/cuts nucleotides from the ends of a nucleic acid molecule. The enzyme removes nucleotide from the 5′ or 3′ end of a DNA molecule. Unlike endonucleases, these never produces internal cuts within DNA molecule. Examples of some exonucleases used in recombinant DNA technology are Exonuclease Bal31, E. coli exonuclease III, Lambda exonuclease, etc

Figure 2 Functioning of exonucleases and endonucleases

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

(c) DNA ligase The function of these enzymes is to ligate or join two fragments of DNA. This is achieved by the synthesis of the phosphodiester bond. In recombinant DNA technology, DNA ligases are used for sealing nicks between adjacent nucleotides. (d) DNA polymerases: DNA polymerases are associated with the synthesis of a new complementary DNA strand of an existing DNA or RNA template. Examples of some DNA polymerases used commonly in genetic engineering are:

 DNA polymerase 1 prepared from E coli.  Taq DNA polymerase used in PCR (Polymerase Chain Reaction).  Reverse transcriptase that uses RNA as a template for synthesizing a new DNA strand (called as cDNA/complementary DNA). Its main use is in the formation of cDNA libraries. (e) Other enzymes

Other enzymes which are also used in rec DNA methods are shown in Table 2.

Table 2 Other enzymes used in recombinant DNA technology

Enzyme Function Terminal deoxynucleotidyl Adds single stranded sequences to 3′-terminus of the DNA molecule. transferase One or more deoxynbonucleotides (dATP, dGTP, dl IP, dCTP) are added onto the 3′-end of the blunt-ended fragments Alkaline Phosphatase Removes phosphate group from the 5′-end of a DNA molecule Polynucleotide Kinase Adds phosphate group to the 5′-end of a DNA molecule

3.3.2 Cloning Vectors:

Cloning vector is a DNA molecule that has the capability to replicate within the host organism. The target DNA is introduced into this vector to produce the recombinant DNA molecule. A great variety of cloning vectors are in use with E. coli which acts as host organism. Sometimes 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. Different types of DNA molecules may be used as cloning vehicles such as they may be plasmids, bacteriophages, cosmids, phasmids or artificial chromosomes

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

Cloning

The process of introducing of a foreign gene (DNA fragment, isolated from a donor organism) into another (vector) is called cloning.

Plasmids

Plasmids are circular double stranded DNA molecules, found mostly inside E.coli. Plasmids are also available commercially.

3.3.3 Host Organism:

A host organism is important because it facilitates the replication of the recombinant DNA molecule. Most widely used host for recombinant DNA technology is E. coli. This is because cloning and isolation of DNA inserts is very easy in this host. A good host organism should be easy to transform and in which the replication of recombinant DNA is easier.

3.3.4 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. It can be of viral, plant, animal or bacterial origin.

3.3.5 Linker and Adaptor Sequences:

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

Both are short, chemically synthesized, double stranded DNA sequences.

 Linkers have (within them) one or more 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. 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 (Figure 3).

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Figure 3 Linker sequences and their role in recombinant DNA technology

 Adaptors are molecules which have been synthesized chemically and designed to possess one or both sticky ends. The foreign DNA is ligated with adaptor on both ends. This new molecule, so formed, is then phosphorylated at the 5′-terminii and then integrated into the vector DNA to form the recombinant DNA molecule (Figure 4).

Figure 4 Adapter sequences and their role in recombinant DNA technology 10

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3.4 Steps in Recombinant DNA technology: Expression of prokaryotic gene

Step1: Identification and isolation of gene/DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically by restriction endonuclease. This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned DNA.

The target gene to be cloned can be obtained from various sources such as genomic library, cDNA library, chemical synthesis of gene or using techniques of gene amplification such as PCR

Step 2: Selection of a suitable cloning vector. Most commonly used vectors are plasmids and bacteriophages. The size of the gene fragments which is to be cloned often determines the selection of vector.

Step 3: Joining of target gene into select vector to construct a recombinant DNA. The target DNA or the DNA insert is ligated (joined) to vector DNA to form a recombinant DNA molecule by DNA ligase. The recombinant DNA molecule is often called as cloning-vector-insert DNA construct (Figure 5).

Figure 5 Use of DNA ligase to form recombinant DNA molecule

Step 4: Introduction of recombinant DNA into a suitable host. The recombinant DNA molecule is introduced into suitable host cells. Selected hosts are bacterial cells like E. coli. Sometimes yeast, fungi may also be utilized. This process of entry of recombinant DNA into the host cell is called transformation.

Recombinant vector can be introduced into host cell by different gene transfer methods

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

a. Physical gene transfer methods:

 Electroporation  Microinjection  Gene transfer mediated by Liposomes  Gene transfer mediated by silicon carbide fibre  Gene transfer mediated by ultrasound  DNA transfer via pollen b. Chemical gene transfer methods:

 Gene transfer mediated by Poly Ethylene Glycol  Gene transfer mediated by calcium Chloride  Gene transfer mediated by DEAE dextran c. Imbibitions of DNA by cells, tissues or organs

d. Gene transfer mediated by virus

Step 5: Selection of transformed host cells with gene of interest from among the non transformed cells

Various methods are employed for separation of the transformed cells (host cells which have taken up the recombinant DNA molecule) from the non-transformed ones. These include antibiotic resistance, use of visible characters, performing assays for biological activity, colony hybridization or blotting test.

Step 6: Expression and Multiplication of target gene in the host:

The selected cells are then cultured in large scale. The inserted gene within the vector that has been transformed will therefore replicate within the host to synthesize multiple copies of the desired gene.

Figure 6 below gives a schematic representation of the various step involved in recombinant DNA technology.

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

Figure 6. Basic steps involved in rec DNA technology

3.5 Bacterial/prokaryotic expression vector

For expression of the desired gene, an expression vector is used.

An expression vector is designed for the expression of the protein which is coded by the cloned gene. It is basically a cloning vector with specific suitable expression signals to facilitate maximum gene expression. The following expression signals are introduced to get maximum gene expression: strong promoter, strong termination codon, transcription termination sequence, translation initiation sequence, adjusting the distance between promoter and cloned gene

When the target gene is cloned into an expression vector and when the expression system is the bacteria, bacterial expression of target protein occurs. This is also known as prokaryotic expression.

Given below in Figure 7 is a simple representation of an expression vector (for e,g, plasmid). It is however more complex in reality.

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

Figure 7 Typical prokaryotic expression vector

The figure shows a typical prokaryotic expression vector, with a cloned gene of interest. An origin of replication, a promoter and a ribosomal binding site are positioned upstream of the target gene that has been cloned into this prokaryotic expression vector. Additionally, one or more transcriptional termination sequences are positioned downstream.

The selection marker commonly used is a drug-resistance gene encoding an enzyme that inactivates a specific antibiotic and helps in selection of transformed cells over non-transformed ones. When the transformed bacteria is exposed to a specific antibiotic, only those that have been able to successfully take up the recombinant DNA/plasmid will have survived, since the expression of the antibiotic resistance gene in the plasmid will have made them resistant to the antibiotic.

An example of prokaryotic expression vector is pET-3a vector (Figure 8). The host for this plasmid is BL strain of E. coli.

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology

Figure 8 pET-3a vector

The product is synthesized in mass cultures in large quantities. This is how insulin is produced in large quantities in cell cultures

3.5.1 Advantages of prokaryotic expression system:

 The prokaryotic system allows for obtaining large quantities of recombinant proteins in a short time.  Culturing of bacteria is simple and inexpensive.  The simplicity of genetic modifications and the availability of many bacterial mutants. 4. Summary In this lecture we learnt about:

 Elements of recombinant technology  Steps in recombinant DNA technology  The prokaryotic gene expression  Advantages of prokaryotic gene expression system

Biochemical Techniques Biochemistry 26 Recombinant DNA Technology