BIOTECHNOLOGY. Sticky & blunt ends. Restriction endonucleases. Gene cloning an overview. DNA isolation & restriction

Transcription

1 BIOTECHNOLOGY RECOMBINANT DNA TECHNOLOGY Recombinant DNA technology involves sticking together bits of DNA from different sources. Made possible because DNA & the genetic code are universal Biology Olympiad Preparation Program Biology Olympiad Preparation Program 3 Restriction endonucleases Bacteria use restriction enzymes to cut foreign DNA. Restriction enzymes are specific for a particular restriction site. Restriction fragments are produced by digesting DNA with a particular restriction enzyme. EcoRI Escherichia coli, strain R, enzyme 1. AluI Sticky & blunt ends Sticky ends must be compatible for complementary base pairing. - C T T A A - G - C T T A A - G G - A A T T C - G - G C A T C - Blunt ends do not have this requirement. - A G - T C C T G A Biology Olympiad Preparation Program 4 - A G - T C T T A A Biology Olympiad Preparation Program 5 RE Grow bacteria Gene cloning an overview Isolate plasmid DNA from bacteria Mix & ligate fragments with DNA ligase Isolate human DNA with gene of interest Transform bacteria Isolate bacterial clone carrying gene of interest same RE amp R DNA isolation & restriction lacz Restriction site same RE Gene of interest 2004 Biology Olympiad Preparation Program Biology Olympiad Preparation Program 7 1

2 Ligation of DNA Transformation Mix DNA ligase Transformation 2004 Biology Olympiad Preparation Program Biology Olympiad Preparation Program 9 Gene cloning Cell cloning & identification Plate out bacteria on solid nutrient media Only transformed bacteria can grow Ampicillin + X-gal White colonies have human DNA inserts pick these Β-galactosidase hydrolyses X-gal into a blue substance Colonies with disrupted lacz genes appear white 2004 Biology Olympiad Preparation Program Biology Olympiad Preparation Program 11 Clone identification Complementary DNA Transfer some cells to filter Wash filter with probe Are we able to take a gene straight from a human and expect bacteria to express it (make proteins from it) properly? Lyse cells & Denature DNA Autoradiography Eukaryotic genes have introns which prokaryotes cannot splice. The protein product would most likely be incorrect. DONE! 2004 Biology Olympiad Preparation Program 12 Hence the importance of cdna if we want to express eukaryotic genes in bacteria Biology Olympiad Preparation Program 13 2

3 DNA ANALYSIS & PRACTICAL APPLICATIONS DNA libraries Since we can cut, copy and paste DNA, we can make libraries of different DNA fragments. Fragments of kangaroo DNA in books of library 2004 Biology Olympiad Preparation Program 14 Plasmid library Phage library 2004 Biology Olympiad Preparation Program 15 Primers anneal Denature DNA DNA polymerase adds nucleotides to make new DNA strands 2 strands after 1 st cycle 2004 Biology Olympiad Preparation Program Biology Olympiad Preparation Program 17 Takes just 30 cycles to make over 1 billion copies of the DNA. PCR is fast, efficient and specific. Specificity determined by the primers used. PCR is a technique widely used to amplify DNA from a wide variety of sources. Gel electrophoresis Electrophoresis separates macromolecules (DNA, proteins) according to their charge and size. DNA has phosphate groups, making it negatively charged. DNA is run on an agarose gel which provides a solid, porous medium for the molecules to move through. Wells Only requires a small amount, sometimes just a single cell, of starting material Biology Olympiad Preparation Program Biology Olympiad Preparation Program 19 3

4 Southern blotting Southern blots allow the detection of particular DNA sequences. Northern & Western blotting Northern blots detect and analyse RNA. Western blots detect and analyse protein. Both use the same principle as Southern blotting i.e. electrophoresis, transferral to filter, probe binding Biology Olympiad Preparation Program Biology Olympiad Preparation Program 21 Restriction fragment length polymorphisms Person A: RFLPs allow DNA comparison of different individuals. 3 fragments electrophoresed: DNA fingerprinting A DNA fingerprint is a specific pattern of bands that is essentially unique to the individual. These bands can be obtained by analysing RFLPs. Person B: 2 fragments electrophoresed: Detectable difference between length of restriction fragments, which are dissimilar (polymorphic) between individuals Biology Olympiad Preparation Program Biology Olympiad Preparation Program 23 DNA fingerprinting Forensics DNA fingerprints are now accepted as compelling evidence by legal experts and scientists. Probability of 2 people having identical DNA fingerprints is between 1/100,000 and 1/1 billion. Who does the bloodstain belong to? 2004 Biology Olympiad Preparation Program Biology Olympiad Preparation Program 25 4

5 Sanger sequencing Sanger sequencing DNA polymerase cannot move past a ddntp. + dntps + ddatp A T A T T A C G Etc 2004 Biology Olympiad Preparation Program Biology Olympiad Preparation Program 27 Modern DNA sequencing Fragments run on a single lane Chromosome maps Computers can piece together DNA sequences into a whole chromosome map bioinformatics. Colours read by laser Original sequence deduced Genomics the study of genomes based on their DNA sequences. Genome organisation. Control of gene expression. Growth & development. Evolution Biology Olympiad Preparation Program Biology Olympiad Preparation Program 29 Plant gene technology - Agrobacterium Ti plasmid Agrobacterium Gene of interest Agrobacterium causes crown gall disease in plants it infects by introducing its Ti plasmid into the plant cell. Plant gene technology gene gun DNA coated onto tiny gold or tungsten particles Pellets are placed inside a gene gun Allow recombinant Agrobacterium to infect plant cell Transformed plant cell Tissue culture Mature plant 2004 Biology Olympiad Preparation Program 30 Pellets are shot into plant tissue Some pellets will enter the nucleus, and some foreign DNA will successfully incorporate into the plant cell genome Biology Olympiad Preparation Program 31 5