Recombinant DNA Technology Lecture PDF

Recombinant DNA Technology Lecture 12 What is Recombinant DNA? • Recombinant DNA technology, also called gene cloning, genetic engineering, or molecular cloning, is an umbrella term encompassing a number of protocols which allows DNA to be produced via artificial means • Recombinant DNA (rDNA) is...

Recombinant DNA Technology Lecture 12 What is Recombinant DNA? • Recombinant DNA technology, also called gene cloning, genetic engineering, or molecular cloning, is an umbrella term encompassing a number of protocols which allows DNA to be produced via artificial means • Recombinant DNA (rDNA) is constructed with DNA from different sources. 1. This is made possible by two important enzymes: Restriction Enzymes and DNA Ligase 2. A vector - A vehicle to transfer foreign genetic material into another cell. 3. A Host cell - organisms that are used to carry out the genetic modification Basic Principle of rDNA • DNA is extracted from a source organism and cleaved enzymatically. • Each enzymatically cut DNA fragment (insert DNA) is joined (ligated) to a cloning vector (Choice of vector depends on size of insert and host to be infected) • Each DNA construct is transferred into a host cell, where it is maintained. • Screening/identification of the host cell colonies containing the rDNA molecules (i.e. screening the ‘library’ of clones generated) in order to identify the specific colony containing the target DNA fragment, i.e. the target gene Restriction Endonucleases • Both the source DNA that contains the target sequence and the cloning vector must be consistently cut into discrete and reproducible fragments – not randomly • Type II restriction endonucleases or restriction enzymes are bacterial enzymes that cut DNA molecules internally at specific base pair sequences. • Found firstly in the bacterium Escherichia coli (E.coli) Naming of Restriction endonucleases • Restriction enzymes are named based on the bacterial species from which they were first isolated: EcoRI How it works? • The restriction enzyme binds to a DNA region with a specific palindromic sequence - the two strands of the binding site are identical when either is read in the same polarity • The EcoRI recognition sequence consists of 6 bp and is cut between the guanine and adenine residues on each strand • In addition to EcoRI, hundreds of other type II restriction endonucleases with more than 120 different recognition sites have been isolated from various bacteria. Sticky Ends • Some restriction enzymes digest DNA, leaving 5’ phosphate extensions; some leave 3’ -hydroxyl extensions. • the overhangs can stick together by complementary base pairing. For this reason, enzymes that leave single-stranded overhangs are said to produce sticky ends. • Sticky ends are helpful in cloning because they hold two pieces of DNA together so they can be linked by DNA ligase. Blunt Ends • Blunt ends are created when a restriction endonuclease cuts both strands of the DNA molecule at the same position, in a symmetrical manner. This results in two DNA fragments with blunt ends that have no unpaired bases • They cannot be easily ligated together without additional steps, such as filling in the overhangs with DNA polymerase or adding linkers with compatible ends to the fragments. The ligation reaction is less efficient and more likely to fail. • Used in specific techniques Protection mechanism in host cells • Under natural conditions, bacteria use restriction endonucleases to cleave foreign DNA and have developed systems that protect their own DNA from being degraded. Most often, methylation of the cytosine residues of a restriction endonuclease site in the host DNA prevents restriction endonucleases from cutting at these sites but leaves the corresponding nonmethylated sites of foreign DNA vulnerable to attack A vector is a DNA molecule that is used to carry a foreign DNA into the host cell. It can selfreplicate and integrate into the host cell. Cloning Vectors Vectors can be a plasmid from the bacterium, a cell from the higher organism or DNA from a virus. The target DNA is inserted into the specific sites of the vector and ligated by DNA ligase. The vector is then transformed into the host cell for replication. Features of Cloning Vectors • origin of replication (ori):When a vector with a foreign DNA insert is introduced into a host cell, such as a bacterium or a yeast cell, the host cell's replication machinery recognizes the origin of replication sequence within the vector and initiates DNA replication from that point. • Restriction Site: for the insertion of the target DNA. • Selectable marker and or Antibiotic resistance gene facilitates screening of the recombinant organism. • Promoter: allows for the controlled expression of the gene of interest in the host cell • multiple cloning sites: to allow for multiple target insertions • It should be small so that it can easily integrate into the host cell. • It should be capable of inserting a large segment of DNA. • It should be capable of working under the prokaryotic and eukaryotic systems. Types of Cloning Vectors: Plasmids • These were the first vectors used in gene cloning. • extrachromosomal circular DNA molecules. These are found in bacteria, eukaryotes and archaea. • They are small in size • Replicate independent of the host • High copy number • Easy detection due to antibiotic-resistant genes. • Small insert size – large fragments cannot be cloned • Standard methods of transformation are inefficient • Examples: pBR322, pUC18 Bacteriophage (viruses that infect and replicate only in bacterial cells.) • Contain MCS • Larger insert size than plasmids • Presence of cos sites - short complementary single strand extensions that anneal upon entry of the viral DNA into a host cell. Annealing circularizes the viral DNA. • The screening of phage plaques is much easier than the screening of recombinant bacterial colonies. • Examples: Phage λ and M13 Cohesive sites (cos sites) Cosmid (modified plasmid) • Hybrid vectors composed of plasmid and λ-phage vectors • Cosmids are similar in structure to plasmids, but they contain a bacteriophage cohesive terminal site (cos), which allows them to be packaged into bacteriophage particles and transferred into bacterial cells via infection. • Large DNA fragments (45kb) • High transformation efficiency, producing large number of clones • Example: pHC79 BACs and YACs Bacterial Artificial Chromosomes (BACs) Yeast Artificial Chromosomes (YACs) A DNA construct based on a functional fertility plasmid (F plasmid Vector constructed from the telomeric, centromeric and replication origin sequences needed for replication in yeast cells Transformed into Bacteria Transformed into Yeast and bacteria Circular Linear 1-2 vectors occur per bacterial cell (low yield) 1 vector occurs per yeast cell (lower yield) Up to 300 kb insert size Up to 2000 kb insert size DNA inserts with repetitive sequences are unstable in BACs and can be deleted and rearranged Human Artificial Chromosomes • No upper limit on DNA that can be cloned • Avoids possibility of insertional mutagenesis • Utilised for gene transfer into human cells Insertion of Recombinant DNA Into Host. • In this step, the recombinant DNA is introduced into a recipient host cell. • Once the recombinant DNA is inserted into the host cell, it gets multiplied and is expressed in the form of the manufactured protein under optimal conditions. Host Cell Choice 1. Bacteria are often used because they are easy to manipulate, grow quickly, and have a high replication rate. 2. Yeast cells are used because they can produce large amounts of protein. 3. Mammalian cells are used when the desired product is a protein that needs to be post-translationally modified. Insertion into Host Introducing foreign genetic material into cells Transfection Transformation Transduction involves the introduction of rDNA into eukaryotic cells It involves the uptake and incorporation of rDNA into the bacterial cell. This process can occur naturally in some bacteria or can be induced in the laboratory. is transferred from one bacterium to another through a viral vector Cell Competency • Treatment of host cells with calciumchloride makes the cells more permeable to take up exogenous DNA. This cell stage is called competent cells. Then, pores are created on the cell membrane temporarily by a heat shock. • In electroporation, cells are made competent by giving an electric shock. Recombinant Insulin • Prior to the development of recombinant DNA technology, insulin was extracted from the pancreas of animals, which was a time-consuming and expensive process. • Recombinant DNA technology has allowed to produce human insulin using bacteria as host cells. This has made insulin more widely available and affordable for patients with diabetes. Recombinant human growth hormone (hGH) • hGH plays a crucial role in growth, development, and metabolism (hypopituitary) • hGH was extracted from cadaveric pituitary glands. • In 1985, an epidemic of Creutzfeldt–Jakob disease, caused by contamination of pituitary-derived hGH with the prion protein, caused the deaths of more than 200 children worldwide. • Cloning and expression of hGH in Chinese hamster ovary (CHO) cells. This allowed for the production of large quantities of pure and uncontaminated hGH. The use of mammalian cells ensured proper post-translational modifications, such as glycosylation for proper functioning.