8.1 R DNA Technology

Dr V Malathi

8 Biotechnology- Biology for Human Welfare

8.1 R DNA Technology

R DNA Technology

Dr V Malathi

What is r DNA ?

Recombinant DNA molecules are hybrid DNA molecules formed by joining the DNA sequences /segments from varied sources . Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is
incorporated into a single recombinant molecule. Genetic engineering involves the use of recombinant DNA technology.
Molecular cloning is a set of methods used to construct recombinant DNA and incorporate it into a host organism.
If the DNA that is introduced comes from a different species, the host organism is now considered to be transgenic.

Applications of r DNA

Recombinant DNA (rDNA) is widely used in biotechnology, medicine and research
Recombinant DNA technology has been used to produce various human proteins in microorganisms
• Examples of products of recombinant DNA technology in medicine and research include: human recombinant insulin, growth hormone, blood clotting factors, hepatitis B vaccine, diagnosis of HIV infection.
• Examples of products of recombinant DNA technology in agriculture include: herbicide-resistant crops, and insect-resistant crops.

What are cloning Vectors ?

Cloning vectors are vehicles that are used to introduce foreign DNA into host cells, where that DNA can be reproduced (cloned) in large quantities.
• Examples of cloning vectors are : plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast
artificial chromosomes (YACs).

What are Tools of r DNA Technology?

Recombinant DNA requires 3 key molecular tools:
1.Cutting DNA at specific sites – most often performed by enzymes called restriction endonucleases (restriction enzymes).
• Restriction enzymes often make staggered cuts at specific 4, 6, or 8-bp palindromic sequences in duplex DNA, leaving characteristic “sticky ends” that can anneal to each other via hydrogen bonding between complementary bases on the single-stranded overhangs.
2.Ligating DNA fragments with an enzyme called DNA ligase.
• DNA ligase, creates covalent phosphodiester bonds between any two DNA fragments that have been cut by the same restriction enzyme, or have the same compatible “sticky ends”.
3.A “vector”, such as a plasmid, that can be used to insert a new segment of DNA via restriction enzyme cutting and ligation. The plasmid containing the inserted DNA segment will replicate in host cells.

What are steps to construct a r DNA?

“Recombinant DNA” by Angela N.H. Creager,creativecommons.org via Wikimedia Commons is licensed under CC BY 4.0

Step 1: Isolation of Gene of interest

Gene of interest is first isolated. For this, initially the cells containing the gene of interest is isolated and disrupted to release nucleus. From the nuclear fraction, the gene of interest is released by using the restriction enzyme which posses the appropriate restriction sites at both ends of the gene of interest. After the gene of interest fragmented, they are separated by using normal isolating procedures like electrophoresis or chromatography. Building a DNA library, which is an extensive collection of cloned DNA fragments from a cell, tissue, or organism, is frequently the first step in isolating a particular gene.

Step 2 : Selection of suitable Vector
The function of the vector is to enable the foreign genes to get introduced into and become established within the host cell. Naturally occurring DNA molecules that satisfy the basic requirements for a vector are plasmids and the genomes of bacteriophages and eukaryotic viruses. They are further classified as cloning and expression vectors depending on the stage of genetic engineering at which these vectors are used. Many bacteria contain extra-chromosomal DNA elements called plasmids. These are usually small (a few 1000 bp), circular, double stranded molecules that replicate independently of the chromosome and can be present in high copy numbers within a cell. Plasmids can be used as Cloning Vectors

Step 3 : Creating a r DNA- Restriction Digestion & Ligation
To insert a DNA fragment into a plasmid, both the fragment and the circular plasmid are cut using a restriction
enzyme that produces compatible ends. Restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome).Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand.
A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the
palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends) while others cut without generating such overhangs, instead producing blunt ends.After restriction digestion, the desired fragments may be further purified or selected before they are mixed together with ligase to join them together.
Following a short incubation, the newly ligated plasmids, containing the gene of interest are transformed into a
suitable host.

Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA.
Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugarphosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand.
The ligase enzymes of E. coli and phage T4 have the ability to seal the single stranded nicks between nucleotides in a duplex DNA.

Step 4: Transformation in to host

Transformation is accomplished by mixing the ligated DNA with host cells e.g., E. coli cells that have been specially
prepared (i.e. made competent) to uptake DNA. Competent cells can be made by exposure to compounds such as
CaCl2 or to electrical fields (electroporation)

Step 5: Selection of Transformed cells
Only a small fraction of cells that are mixed with DNA will actually be transformed,
Directional selection
The phenotypes conferred by the cloned genes on the host are used as markers of selection. All useful vector molecules carry a selectable genetic marker or have a genetically selectable property. Plasmid vectors generally possess drug resistance or nutritional markers and in phage vectors the plaque formation itself is the selectable property

Insertional inactivation
The technique depends upon homologous recombination between DNA cloned and the host genome. If the cloned sequence lacks both promoter and sequences encoding essential regions of the carboxyl terminus of the protein, recombination with homologous genomic sequences will cause gene disruption and produce a mutant genotype.
On the other hand, if the cloned fragment contains appropriate transcriptional and translational signals, homologous recombination will result in synthesis of a functional mRNA transcript, and no mutant phenotype will be observed

Step 6 :Expression of cloned genes
An expression vector, otherwise known as an expression construct. It is usually a plasmid or virus designed for gene expression in cells.
The vector is used to introduce a specific gene into a target cell .It can control the cell’s mechanism for protein synthesis to produce the protein encoded by the gene. Therefore in addition to the gene of interest, these expression constructs also contain regulatory elements like enhancers and promoters so that efficient transcription of the gene of interest occurs.

Step 7 : Collection & Purification of Recombinant proteins
As the recombinant proteins are produced by the cloned genes, they start accumulating. The next task is to collect and purify the specific gene product i.e., the requisite protein. This is not an easy job since many a times the recombinant protein is foreign to the host cell which possesses an enzyme machinery to degrade the outside proteins.

The yield of production of recombinant proteins is efficient if they are quickly exported and secreted into the environment (surrounding medium). Further, the recovery and purification of foreign proteins is easier from
the exported proteins. Serious efforts have been made to develop methods for increasing the export of recombinant proteins. Some of the species of the bacterium, Bacillus subtilis normally secrete large quantities of extracellular proteins. A short DNA sequence, called signal sequence from such species is introduced into other B. subtilis. These bacteria produce recombinant DNA tagged with signal peptide, which promotes export and secretion. The signal peptide can be removed after purification of foreign protein.