Recombinant DNA Technology Lecture 2 PDF

BIOTECHNOLOGY: RECOMBINANT DNA TECHNOLOGY References: LECTURE 2 Russell et al. 4th edition pp326-329, pp408-415...

BIOTECHNOLOGY: RECOMBINANT DNA TECHNOLOGY References: LECTURE 2 Russell et al. 4th edition pp326-329, pp408-415 Russell et al. 5th edition pp315-321, pp388-395 Class notes Key concepts: Central dogma, restriction enzymes and ligase, PCR, plasmid vectors, libraries Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY THE CENTRAL Flow of information from DNA (genotype) to protein (phenotype) DOGMA Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY DNA POLYMERASE : Replication = process where identical DNA molecules are made THE CENTRAL RNA POLYMERASE : Transcription = process where RNA is made using DOGMA DNA as a template RIBOSOME: Translation = process where protein is made using mRNA Start codon REPLICATION 5’ 3’ ’ 3’ 5’ Double ’ ’ stranded 5’ 3’ Single stranded Stop codon Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY Gene structure: THE CENTRAL A gene consists of regulatory domains (promotor, activator-, DOGMA silencer- and terminator sequences) and an open reading frame (ORF) that contains exons and introns ATG  Only 1 universal start codon for all organisms Activator Silencer Promotor ORF Terminator ATG When? What? Stop! Where? Prokaryotic genes are How much? simpler with no introns Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY HOW CAN WE MANIPULATE DNA? Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY Bacterial restriction enzymes cut DNA molecules at specific DNA USING RESTRICTION sequences called restriction sites ENZYMES, DNA A restriction enzyme usually makes many cuts, yielding restriction LIGASE AND PLASMIDS fragments The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY DNA ligase is an enzyme that seals the bonds between restriction USING RESTRICTION fragments ENZYMES, DNA LIGASE AND PLASMIDS Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY WHAT IS A PLASMID? Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY Plasmid Component Description Origin of Replication (ORI) DNA sequence where plasmid replication starts DIFFERENT COMPONENTS OF Antibiotic Resistance Gene Allows selection of bacteria that carry the plasmid A PLASMID Short DNA sequence containing many unique restriction Multiple Cloning Site (MCS) enzyme cutting sites to allow different options for cloning the DNA of interest into the plasmid.. Piece of DNA that has been ligated into the plasmid, Insert normally in the MCS Drives transcription of the insert DNA, leading to the production of recombinant protein. Promoters can lead to Promoter Region constitutive expression, expression at certain growth phases, or be induced by chemicals Allows selection of an organism that contains that price of DNA. In bacteria it would normally be and antibiotic Selectable Marker resistance gene (as above). In plants it could also be a herbicide resistance gene. Short piece of DNA where synthesised DNA primers can Primer Bindings Sites bind, either to allow sequencing of the DNA or to amplify by the polymerase chain reaction. Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY DIFFERENT COMPONENTS OF A PLASMID Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe  Restriction site RECOMBINANT DNA DNA 5′ 3′ Fig. 20-3-1 3′ 5′ 1 Restriction enzyme USING cuts sugar-phosphate backbones. RESTRICTION ENZYMES, DNA LIGASE AND Sticky end PLASMIDS 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination 3 DNA ligase seals strands. Recombinant DNA molecule Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY DNA-CLONING Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY One problem with cloning is the identification of bacteria PROBLEM... containing plasmids with an insert. An antibiotic can select for a bacterium that contains a plasmid, BUT… How can you tell if a bacterium contains a plasmid with an insert or not? Add ampicillin Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY α-complementation PROBLEM... Some E. coli strains have modified lacZ with lacZ’ region deleted SOLVED Encodes first 146 amino acids of β-galactosidase LacZ’ is placed into the cloning vector The other half of the lacZ gene is present in the chromosome. These strains only express a functional enzyme if they harbour a plasmid which carries the lacZ’ region LacZ’ encodes part of β-gal Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY PROBLEM... SOLVED BLUE - plasmid containing NO insert Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY WHERE DOES A number of sources, but mostly from: THE DNA COME FROM? Polymerase Chain Reaction (PCR) Only works if you know what the sequence of the DNA is. Libraries Can be used to identify genes based on DNA similarity to other genes Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe IN VITRO AMPLIFICATION OF DNA Polymerase chain reaction (PCR) PCR components and process DNA template Primers to delimit the fragment to be amplified DNA polymerase The 4 dNTPs (G, A, T and C) Hybridization is very specific between complementary sequences: 5’-ATTGCGTACTAGCATGA-3’ 3’-TAACGCATGATCGTACT-5’ So, specific sequences can be identified Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe using primers. RECOMBINANT DNA TECHNOLOGY 5′ 3′ TECHNIQUE Target sequence Genomic DNA 3′ 5′ THE POLYMERASE 1 Denaturation 5′ 3′ CHAIN REACTION (PCR) Annealing 2 3′ 5′ Primers Cycle 1 yields 2 molecules 3 Extension New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY Thermocycler THE POLYMERASE BUT: CHAIN REACTION For PCR to work you need to know the DNA (PCR) sequence. If you don’t, then the best way is to make a library. Photocopy PCR The book The whole genome The pages and The piece of DNA you want to amplify and the bookmark primers you will use Paper and ink The 4 bases (ATCG) that make up DNA The photocopier PCR Machine Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY TYPES OF DNA Many cloning strategies begin by preparing a DNA library LIBRARIES Libraries are collections of cloned DNA fragments from a particular source contained within bacteria or viruses as the host Libraries can be saved for relatively long periods of time and screened to select out different genes of interest Three types of libraries are typically used for cloning Genomic DNA libraries Metagenomic libraries Complementary DNA (cDNA) libraries Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY TYPES OF DNA Type of Library Description Contains randomly cut pieces of LIBRARIES DNA isolated from the genome. If it Genomic is from a eukaryote it contains both exons and introns Contains randomly cut pieces of DNA isolated from an Metagenomic environmental sample Contains DNA synthesised from RNA using the enzyme reverse cDNA transcriptase. Contains no introns. Reverse transcriptase Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe PRODUCTION OF A GENOMIC DNA LIBRARY Restriction WHAT IS IT? enzyme digestion. E.g. EcoRI A library from the gDNA of an organism (ALL the DNA in the genome) DNA fragment unknown? Isolate DNA and fragment using restriction enzymes Ligation with DNA ligase Ligate fragments into plasmids Same concept as Recombinant DNA technology Bacteria take up plasmids Millions of individual DNA fragments will be ligated with insert into plasmids Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe  TECHNIQUE Hummingbird cell Bacterial cell lacZ gene Restriction Sticky Gene of interest site ends TECHNIQUE ampR gene Bacterial plasmid Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying non- Colony carrying recombinant recombinant plasmid plasmid with disrupted lacZ gene with intact lacZ gene One of many bacterial clones Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY DNA can be used for recombinant protein production: APPLICATIONS Protein hormones Enzymes for washing powders Vaccine epitopes Molecular biology reagents.... Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe RECOMBINANT DNA TECHNOLOGY DNA metagenomic library made at the IPB APPLICATIONS produces interesting polymers... Where did the DNA in the libraries come from? Levan polymer (Levan-sucrase enzyme) Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe Bg32 polymer (Bg32-β-galactosidase enzyme) RECOMBINANT DNA TECHNOLOGY NEXT TIME WE Artificial LIFE WILL DISCUSS: RECOMBINANT DNA TECHNOLOGY Part II Science | EyeNzululwazi ngezeNdalo | Natuurwetenskappe