Recombinant DNA Technology Lecture Notes PDF

Lec 2 Recombinant DNA Technology Ohoud M. Marie MD, PhD – Biochemistry Bioinformatics  Recombinant DNA Technology Recombinant DNA technology also called gene cloning, genetic engineering, or molecular cloning, is an umbrella term encompassing a number of proto...

Lec 2 Recombinant DNA Technology Ohoud M. Marie MD, PhD – Biochemistry Bioinformatics  Recombinant DNA Technology Recombinant DNA technology also called gene cloning, genetic engineering, or molecular cloning, is an umbrella term encompassing a number of protocols.  Recombinant DNA Technology the purpose of these experimental manipulations is to introduce a gene from one organism into a host cell, where it can be perpetuated and studied.  Recombinant DNA Technology DNA is extracted from a source organism and cleaved enzymatically. Each enzymatically cut DNA fragment (insert DNA) is joined (ligated) to a specifically designed DNA entity (cloning vector), capable of replicating in a particular host cell, to form an intact recombinant DNA molecule (DNA construct). Each DNA construct is transferred into a host cell, where it is maintained. Usually, a host cell receives only one DNA construct. However, the efficiency of uptake of DNA constructs is very low.  Recombinant DNA Technology Consequently, the host cells that carry DNA constructs must be identified and separated (selected) from those that do not. Moreover, the host cell with a specified DNA insert must be recognized and isolated. Subsequently, the insert DNA (target DNA, cloned DNA) of a DNA construct can be characterized. In some cases, the insert DNA is expressed in the host cell and, if required, the protein encoded by a cloned gene can be harvested. Schematic representation of DNA cloning. DNA from a source organism (source DNA) is cleaved with a restriction endonuclease and inserted into a cloning vector to form DNA constructs. Each DNA construct is introduced into a host cell. Cells carrying the target DNA construct are identified, isolated, and grown. Restriction Endonucleases  A special class of DNA cutting enzymes that exist in many bacteria Restriction Endonucleases Restriction enzymes were named for their ability to restrict, or limit, the number of strains of bacteriophage that can infect a bacterium.  Restriction Endonucleases Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (4 to 6 bases). These regions are called recognition sequences and are randomly distributed throughout the DNA. Bacteria prevent their own DNA from being degraded by disguising their recognition sequences. Enzymes called methylases add methyl groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease.  Restriction Endonucleases The recognition sequences are usually palindromic, which means that their recognition sequence reads the same in the 5' to 3' direction on both DNA strands.  Restriction Endonucleases There are four different categories of restriction enzymes: Type I Type II cut within or close to their recognition sequence. Type III Type IV Type II restriction enzymes, which cut within their recognition sequence, are the most useful for laboratory experiments.  Restriction Endonucleases Type II restriction enzymes are used to cut DNA molecules at interesting specific locations and then reattach different DNA sequences to each other using an enzyme called DNA ligase, creating new, recombined DNA sequences, or essentially new DNA molecules. This powerful approach to cutting and pasting DNA molecules is known as DNA cloning or recombinant DNA technology.  Sticky Ends The sticky ends join spontaneously by hydrogen bonding (base pairing).  Blunt Ends Both strands of DNA are cut in the same place.  If two fragments of DNA from different sources have been produced by the action of the same restriction enzyme, the two pieces will have identical sets of sticky ends and can be spliced (recombined) in vitro. The enzyme DNA ligase is used to covalently link the backbones of the DNA pieces, producing an recombinant DNA molecule (rDNA). Plasmids and Vectors  Plasmids =Small, circular, double-stranded DNA molecule =Distinct from a cell’s chromosomal DNA =Extra-chromosomal genetic elements which aren’t connected to the main bacterial chromosome and they replicate independently of chromosomal DNA =Found mainly in bacteria  Plasmids The genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance. Plasmids have a wide range of lengths, from roughly one thousand DNA base pairs to hundreds of thousands of base pairs.  Synthetic plasmids (Vectors) A vector, in molecular biology, refers to a plasmid that is engineered to make it a more useful tool for molecular biologists The most important property is self-replication; once in a cell, a vector must be capable of replicating Any DNA that is inserted in the vector will be replicated in the process Thus, vectors serve as vehicles for the replication of desired DNA sequence  Characteristics of synthetic plasmids Laboratory-designed plasmids contain a small number of genes that help transformation. These include: = An origin of replication (ori) which is specific sequence of nucleotides where DNA replication begins =A multiple cloning site which contains a recognition sites for specific restriction enzymes. These restriction enzymes can be used to cut the plasmid, so foreign DNA can be pasted in by ligation  Characteristics of synthetic plasmids =A resistance gene which code for a protein needed by the bacteria in order to survive in a particular growth medium (ex, when a specific antibiotic is present) =Marker genes: common selectable marker genes are for antibiotic resistance or for an enzyme that carries out an easily identified reaction  Shuttle vectors Some plasmids are capable of existing in several different species. They are called shuttle vectors and can be used to move cloned DNA sequences among organisms Shuttle vectors can be very useful in the process of genetically modifying multicellular organisms (ex, by trying to insert herbicide resistance genes into plants To be continued next lecture..