Recombinant DNA Technology

(Introducution)

Dr. Abhishek RoyJunior Resident (III)

Dept. of BiochemistryGrant Govt. Medical College

And Sir JJ Group of Hospitals, MumbaiEmail: mail@abhishek.ro

Twitter: @abhishekroy

Objectives

Concept of Recombinant DNA Technology. Role of Restriction Endonucleases Role of Various Enzymes used in Recombinant DNA Technology Cloning Vectors types, purpose and method of preparation

Plasmids (pBR322) BAC YAC

What is RDT? A clone is an identical copy. The process DNA Cloning involves

Cutting target DNA at precise locations. Selecting a small carrier molecule of DNA capable of self-replication. Joining two DNA fragments covalently. Moving recombinant DNA from the test tube to a host cell. Selecting or identifying host cells that contain recombinant DNA.

The methods used to accomplish these and related tasks are collectively referred to as recombinant DNA technology or, more informally, genetic engineering.

Role of Restriction Endonucleases

Restriction endonucleases (also called restriction enzymes) recognize and cleave DNA at specific sequences (recognition sequences or restriction sites) to generate a set of smaller fragments.

Restriction endonucleases are found in a wide range of bacterial species.

In the host cell’s DNA, the sequence that would be recognized by its own restriction endonuclease is protected from digestion by methylation of the DNA, catalyzed by a specific DNA methylase.

The restriction endonuclease and the corresponding methylase are sometimes referred to as a restriction modification system.

Three types: Type I, Type II and Type III. Type I and Type III require ATP and cleave at a site downstream to the

recognition site

Types of Restriction Endonucleases II

• They recognize palindromic sequences• Restriction Endonuclease II cleave at the recognition site.• Don’t require ATP.• Restriction enzymes are named after the bacterium from• which they are isolated.• The foreign DNA that is cleaved is said to be restricted.

Nomenclature If we take the example of EcoRI or EcoRII

E- Genus name, here Eschericia co- Species name, here coli R- Strain name I/II- Roman numeral to indicate order

of discovery.

Some Enzymes used in Recombinant DNA Research

Digestion with a restriction endonuclease can result in the formation of DNA fragments with sticky, or cohesive, ends (A), or blunt ends (B); phosphodiester backbone, black lines; interstrand hydrogen bonds between purine and pyrimidine bases.

Results of restriction Endonuclease digestion

Cleavage of DNA molecules by restriction endonucleasesa) sticky ends (with protruding single strandsb) Blunt ends.

Blunt ends are annealed directly.• Two alternatives:

• Use Terminal Transferase to add Poly d(G) and Poly d(C) to Vector DNA and Foreign DNA respectively (Homopolymer Tailing)

• Use synthetic blunt-ended duplex oligonucleotide linkers containing the recognition sequence for a convenient restriction enzyme sequence are ligate to the blunt-ended DNA.

• Once a DNA molecule has been cleaved into fragments, a particular fragment of known size can be partially purified by agarose or acrylamide gel electrophoresis or by HPLC.

• After the target DNA fragment is isolated, DNA ligase can be used to join it to a similarly digested cloning vector—that is, a vector digested by the same restriction endonuclease

DNA ligase catalyzes the formation of new phosphodiester bonds in a reaction that uses ATP or a similar cofactor.

The base pairing of complementary sticky ends greatly facilitates the ligation reaction.

Researchers can create new DNA sequences by inserting synthetic DNA fragments (called linkers) between the ends that are being ligated

Inserted DNA fragments with multiple recognition sequences for restriction endonucleases (often useful later as points for inserting additional DNA by cleavage and ligation) are called polylinkers.

Cloning A clone is a large population of

identical molecules, bacteria, or cells that arise from a common ancestor.

This technique is based on the fact that chimeric or hybrid DNA molecules can be constructed in cloning vectors— typically bacterial plasmids, phages, or cosmids. Cloning capacities of common cloning vectors

Use of Restriction Endonucleases for Recombinant or Chimeric DNA molecules

Cloning Vectors The classic E coliThe plasmid pBR322 has

an origin of replication, or ori. The plasmid contains genes that confer

resistance to the antibiotics tetracycline (TetR) and ampicillin (AmpR).

Several unique recognition sequences in pBR322 are targets for restriction endonucleases.

The small size of the plasmid (4,361 bp) facilitates its entry into cells and the biochemical manipulation of the DNA

The constructed E. coli plasmid pBR322

Inserting the DNA Transformation

The cells (often E. coli, but other bacterial species are also used) and plasmid DNA are incubated together at 0ºC in a calcium chloride solution

Subjected to heat shock by rapidly shifting the temperature to between 37ºC and 43ºC.

Electroporation Cells incubated with the plasmid DNA are subjected to a high voltage pulse. This approach, called electroporation, transiently renders the bacterial

membrane permeable to large molecules. Bacteriophage

These are viruses like λ- Phage that can infect bacteria and incorporate its DNA into it.

The DNA of the phage tailored and the phage uses bacterial machinery for replication

Identification of the Cells Regardless of approach relatively few cells take up

plasmids Use of Selectable or Screenable markers.

Selectable markers: Either permit the growth of a cell (positive selection)

or kill the cell (negative selection) under a defined set of conditions. The plasmid pBR322 provides examples of both positive and negative selection

Screenable Markers: Gene encoding a protein that causes the cell to

produce a colored or fluorescent molecule.

Use of pBR322 to clone foreign DNA in E. coli and identifycells containing it

Bacterial Artificial Chromososmes• Plasmid vectors have been develop with special features that allow

the cloning of very long segments (typically 100,000 to 300,000 bp) of DNA

• BAC vectors have stable origins of replication that maintain the plasmid at one or two copies per cell

• BACs also include par genes, which encode proteins that direct the reliable distribution of the recombinant chromosomes to daughter cells at cell division

• The BAC vector includes both selectable and screenable markers.• The BAC vector contains a gene that confers resistance to the

antibiotic chloramphenicol (CmR).• The β-galactosidase catalyzes the conversion of the colorless

molecule 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) to a blue product.

Yeast Artificial Chromosome• YAC vectors contain all the elements needed to maintain a

eukaryotic chromosome in the yeast nucleus: a yeast origin of replication, two selectable markers, and specialized sequences (derived from the telomeres and centromere) needed for stability and proper segregation of the chromosomes at cell division.

• BamHI in removes a length of DNA between two telomere sequences (TEL), leaving the telomeres at the ends of the linearized DNA.

• Cleavage at another internal site EcoRI divides the vector into two DNA segments, referred to as vector arms, each with a different selectable marker.

• DNA fragments of appropriate size (up to about 2 X 106 bp) are mixed with the prepared vector arms and ligated.

• Culture on a medium that requires the presence of both selectable marker genes ensures the growth of only those yeast cells that contain an artificial chromosome with a large insert sandwiched between the two vector arms

Thank You