PhD Course TOPICS IN (NANO) BIOTECHNOLOGY Lecture III 10th April
Overview • So we have looked at what is DNA and what is a gene. • We also looked at DNA replication and protein synthesis, and the path from the gene to protein • This week we will look at Recombinant DNA technology • We will also look at the amplification of DNA and finally at its sequencing
History of Recombinant DNA technology • Antibiotics such as penicillin, the sulfonamides and streptomycin gave much hope • However, in the 50s theye starting to fight back, becoming increasingly resistant to antibiotics • In just a few years 60-80% of bacteria showed resistance not just to one drug, but to multiple drugs • The genes responsible for infectious drug resistance were plasmids, genetic elements that could replicate themselves independently. • In different plasmids, the replication region encodes traits not essential to the bacterial host. • Antiobiotic resistance is one of these traits.
History of Recombinant DNA technology In 1971 Cohen, exploited the antibiotic resistance of the plasmids to selectively enrich offspring that contained cell propogating plasmids. In the late 60s, it was shown that CaCl2 made the cells of E.coli permeable so that they could take up DNA, but could not grow E.coli cells with genetic property changes. In late 1972, Berg reported on methods for joining fragments of DNA outside of cells. Endonucleases, or restrictions enzymes, would however, provide the tool for linking DNA.
Una cerveza y ... In Nov. 1972, Herb Boyer and Cohen met up at a deli bar in Honololu, and discussed the endonuclease that Boyer was working on, and that night they dreamed of the collaborative project that would be the true start of recombinant DNA technology. In March 1973, the pair produced DNA fragments and joined them to plasmids using Boyer’s technique, and then introduced them into bacteria using Cohen’s technique. The first demonstration of DNA cloning had been achieved.
Recombinant DNA technology • The two essential elements of recombinant DNA technology are: • 1. Restriction endonucleases • 2. Vectors for gene cloning
What is a restriction enzyme? • There are two classes of restriction enzymes: • Type I • Cuts DNA on both strands but at non-specific location • Random imprecise cuts • Not very useful for rDNA applications • Type II • Cuts both strands of DNA within the particular sequence recognised by the restriction enzyme
What is a restriction enzyme? • Restriction enzymes (or endonucleases) are bacterial enzymes that cut DNA at very specific sequences • They generally cut in a ‘staggered’ manner, leaving sticky ends but some enzymes generate blunt ends (i.e. Cut DNA in the middle) • Their biological function is to destroy invading foreign DNA
What is a restriction enzyme? • Each bacteria has different restriction enzymes • Enzymes from E.coli cells cut GAATTC/CTTAAG • Enzymes from B. Amyloloquefaciens cut GGATCC/CCTAGG • The restriction enzymes are named after the organism from which they were derived • EcoRI from E.coli • BamHI from B. Amyloloquefaciens
What is a restriction enzyme? • Restriction enzymes are used to make recombinant DNA and gene cloning and genetic engineering were made possible by these enzymes • Over 200 different restriction enzymes are commercially available (some are VERY expensive) • DNA ligase ‘sticks’ the ends back together
What is a restriction enzyme? • Recombinant DNA technology can be used to isolate a genomic clone from DNA or for the isolation of human cDNA • Isolating a genomic clone provides a piece of DNA identical in base sequence to the corresponding stretch of DNA in the cell and is often designed to contain a specific gene • Isolating human cDNA is used for gene expression. Human cDNA (c=complementary) is double stranded DNA copy of mRNA but WITHOUT introns
Vector requirements • Dependent on design of experimental system • Most vectors contain a prokaryotic origin of replication • Antibiotic resistance genes and/or other selectable markers • Examples of cloning vectors are • plasmid • bacteriophages • yeast artificial chromosomes (YAC) • bacterial artificial chromosomes (BAC) • retrovirus
What is a plasmid? • Plasmids are small, extrachromosomal pieces of bacterial DNA that are often antibiotic resistant • They are ‘shuttle vectors’ to create, produce, and maintain recombinant DNA • An example of one of the first plasmids is pBR322 • Both Amp & Tet resistant, Several unique restriction sites • pUC18 now the most commonly used • Derivative of pBR322 • Smaller, Higher copy number per cell, Multiple cloning sites
lacZ gene • Gene encoding for enzyme -galactosidase • Polylinker resides in the middle • Enzyme activity can be measured as marker of gene insertion • Disrupted gene – nonfunctional – WHITE • Intact gene – functional – BLUE • Amp resistance gene still present, Tet resisitance gene omitted
Lambda vector • Bacteriophage lambda () infects E.coli • Double stranded linear DNA vector, suitable for library construction • Can accomodate large segments of foreign DNA, central 1/3 is a ‘stuffer’ fragment • Can be substituted with any DNA fragment of similar size • Can accomodate 15kbp of foreign DNA
Recombinant DNA technology Video 3a: Plasmid Cloning
Genomic Clones • Genes can be cloned in recombinant DNA vectors • Cloning vector • Procedure for cloning a eukaryotic gene in a bacterial plasmid • Isolation of vector and gene-source DNA • Insertion of DNA into the vector • Introduction of cloning vector into bacterial cells • Cloning of cells (and foreign gene) • Identification of cell clones carrying the gene of interest • Nucleic acid hybridization • Nucleic acid probe
cDNA Clones • Genes can be cloned in recombinant DNA vectors • Cloning vector • Procedure for cloning a eukaryotic gene in a bacterial plasmid • Cloning and expression eukaryotic genes: problems and solutions • Difference in promoters • Expression vector • Introns • Complementary DNA (cDNA)
Genomic and cDNA Libraries • Cloned genes are stored in DNA libraries • genomic library – cloned set of rDNA fragments representing the entire genome of an organism • cDNA library - cloned set of rDNA fragments representing genes transcribed in a particular eukaryotic cell type (no introns, extrons etc) • rDNA fragments generated, ligated & cloned • The larger the fragments that are cloned, the smaller the size of the library
Genomic Libraries • Contains at least 1 copy of each fragment • Screened using nucleic acid probes to identify specific genes • Subcloning usually necessary for detailed analysis of genes • N = ln (1-P)/ln (1-f) • e.g. Human genome = 3.2 x 109bp • Lambda vector can accommodate 17kbp inserts • N = ln(1-0.99)/ln(1-(1.7x104bp insert/ • 3.2 x 109bp genome)) • N = 8.22 x 105 plaques required in library
cDNA Libraries • mRNA represents genes that are actively transcribed (or expressed) • Eukaryotic mRNA – introns have been removed • mRNA – converted into a DNA copy (cDNA) • Size of library depends on number of ‘messages’ • More complex than genomic library
ID of specific DNA sequences • Libraries searched using specific probe • Specificity extremely important • Single-stranded nucleic acid fragments • Radioactive vs non-radioactive • Radioisotopes serve as tag - autoradiography • Chemiluminescence, colorimetric, fluorescence • Sources of probes • Heterologous (other species) • cDNA (genomic sequences with introns/promoter elements) • Probe based on protein sequence • 18-21bases sufficient (ssDNA, RNA, antibodies)
ID of specific DNA sequences • Expression Library • Detect protein product of clone using antibodies • Microarray technology • Chromosome walking • If nearby sequences have been cloned, this can be used as starting point for isolation of adjacent genes
Polymerase Chain Reaction • The PCR clones DNA entirely in vitro • Polymerase chain reaction • Denaturation (heat to ~94oC) • Annealing (37-72oC) • Extension (72oC)
Agarose gel electrophoresis • Separation of DNA fragments based on size, charge and shape differences • Standardised MW markers run on the same gel for size comparison
Gel electrophoresis Video
Southern blotting • DNA digested with restriction enzymes and separated by gel electrophoresis • Gel treated with NaOH to denature DNA to ssDNA • DNA transferred from gel to DNA binding filter • DNA ‘fixed’by baking membranes/UV • Incubate with ssDNA probe • Autoradiography/chemiluminescence
Exercises • Describe a plasmid prep. • What is meant by the term ‘sticky ends’? • What is a genomic clone? • What is a cDNA clone? • What are the steps involved in making a genomic library? • Explain in a few sentences the importance & principle of PCR. • Explain in a few sentences the importance & principle of gel electrophoresis. • Explain in a few sentences the importance & principle of southern blotting. • Explain in a few sentences the importance & principle of DNA sequencing.