2/2/16. Insulin and sugar metabolism. A Molecular Genetics Toolbox I: Tools to clone, amplify, analyze and sequence DNA

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1 1. Cold Spring Harbor Labs Learning Centre A Molecular Genetics Toolbox I: Tools to clone, amplify, analyze and sequence DNA 2 Molecular Biology of the Gene (Watson et al.l 6 th (International) Edition 3 8 th Day of Creation. Horace Freeland Judson (Historical) Mani Lecture 2 Insulin and sugar metabolism Type-1 diabetes: Not enough insulin and high blood sugar 1

2 2/2/16 How is insulin produced? From previous lecture(s) Once purified from animal tissue (beef pancreas). Now: recombinant insulin. Identify human gene(s) for insulin. 1. The genome (all our chromosomal DNA) encodes more than 100,000 proteins. 2. A small sequence of DNA encodes each protein. Make bacteria produce mrna and protein Purify the protein DNA cloning, amplification and expression of selected DNA sequences (e.g DNA encoding human Insulin) Package, Store, Q/C, transport. A key concept is of DNA hybridization, based on Watson-Crick base-pair interactions. DNA cloning and in vivo amplification of DNA sequences Key1: Restriction endonucleases (evolved for bacterial defense) Perfectly base-paired helices Remain stable at higher temperatures Key2: Plasmids (self replicating, extra chromosomal circles of DNA). Key3: Bacterial Transformation and growth Werner Arber, Daniel Nathans, Hamilton O. Smith 2

3 ! Restriction Enzymes: site-specific cutting tools for DNA! Restriction Enzymes: site-specific cutting tools for DNA Bacteriophage (e.g. Lambda) Infect and kill/lyse bacteria Phage plaque On E.coli lawn Strain A Plaques Lambda Phage Strain B No Plaques (or occasional rare plaque) Strain B: 1. has a restriction endonuclease that cleaves Phage DNA (eg. EcoR1) at a specific 6-mer 2. A methyl transferase that protects its own genome. Lambda Phage Salvadore Luria Strain A Plaques Strain B No Plaques (occasional, rare plaque) Thanks to an evolutionary arms race there are many different restriction enzymes Salvadore Luria! Restriction Enzymes: site-specific cutting tools for DNA! DNA cloning: amplifying DNA sequences in vivo Restriction enzymes recognize specific short palindromic sequences in DNA: typically 4, 6 or 8 bp The enzyme EcoRI recognizes and cuts GAATTC which occurs on average every ~4.1 kb 1. Extract and purify DNA Chromosome-sized DNA (50 Mb) GAATTC CTTAAG 2X 10 6 kb: We need to purify and grow the ~ 2kb we are interested in. 2. Cut DNA into gene-sized fragments using a restriction enzyme e.g. EcoRI G AATTC CTTAA G single-stranded cohesive ends Insert genomic fragments into a suitable cloning vector Restriction enzymes cut DNA into manageable-sized fragments 3

4 ! Plasmid cloning vectors (viral e.g. lambda-based vectors can also be used) Bacteria have plasmids or episomes (additional chromosomes) with genes conferring antibiotic resistance.! Plasmid cloning vectors Plasmids: small, circular, typically containing the following features: Origin of replication: directs plasmid replication Selectable marker: antibiotic resistance gene Cloning site: a collection of unique restriction enzyme sites Bacteria have restriction enzymes that cut DNA at specific sequences Bacteria and phage also have DNA ligases that stick together/ repair broken DNA Location of restriction enzyme sites for inserting DNA puc19 is a general-purpose cloning vector that accepts DNA inserts of up to 10 kb The lacz region allows blue-white screening using the stain X-Gal Blue colonies contain the original vector (no inserts) Vector puc19 Size: 2.8 kb White colonies contain the vector with an insert that disrupts lacz! Creating recombinant DNA molecules: ligation of vector and genomic fragments! Introduction of recombinant molecules into E. coli (transformation) Plasmid vector puc19 EcoRI Linearized vector DNA Genomic DNA: EcoRI fragments E. coli cells washed in CaCl 2 can take up DNA and be transformed by it Clone Number Cohesive ends of vector anneal with those of random genomic DNA fragment Cells are placed on agar medium containing an antibiotic e.g. ampicillin Extract plasmid DNAs from a colony v T4 DNA ligase seals the gap in the sugar-phosphate backbone Recombinant DNA Individual transformed cells grow to produce visible colonies To check for cloned DNA cut with EcoRI 4

5 ! Genomic libraries: collections of clones corresponding to all sequences in a genome Screen Library Expression vector Human 1000,000 kb genome. From a plasmid library each plasmid housing a 10kb fragment 100,000 colonies corresponds to one genome equivalent (32P DNA probe for insulin DNA) E coli transform and induce How to identify the colony containing insulin coding DNA? Purify Insulin Isolate recombinant insulin in pure form ready for human use (is a little more complicated) Step 1: Clone appropriate constructs Step 2 : E. coli strain 1 produce and purify Chain 1 E coli strain 2 produce and purify Chain 2 Step 3: Mix chains under conditions where appropriate disulfide bonds form 5

6 2. PCR: in vitro amplification of DNA sequences! DNA can be chemically synthesized: oligonucleotides (oligos) Short (21 29 base) single-stranded oligonucleotide of defined base sequence! The availability of oligos (1980s) made the invention of PCR possible Kary Mullis (Nobel Prize 1993) proposed that a pair of oligos could be used to prime the amplification of a target DNA sequence in a cyclical manner Synthetic oligonucleotide primers were key to the development of: Double stranded genomic DNA 1. Separate the DNA strands by heating to 98 o C (a) PCR and (b) DNA sequencing Oligo Primer 1 (forward) 2. Reduce temp to allow oligo primers to anneal (base pair) 3. Reduce temp; Add dntps and DNA polymerase Custom-designed oligos can be ordered on-line from many biotech companies (Eg. Oligo Primer 2 (reverse) Steps 1 3 represent a synthesis cycle Repeat many times 6

7 Morning Glory Pool: Yellowstone the habitat for Thermus aquaticus. PCR uses a heat-stable DNA polymerase from the thermophilic bacterium Thermus aquaticus (Taq DNA pol) This enabled automation A typical PCR amplification cycle PCR machine! Amplification proceeds exponentially PCR and Variations of the basic technique are used for Diagnostics. 1. Chagas outbreaks in his home country inspired Núñez-Mujica to find a cheap, portable PCR machine to help diagnose those infected with Chagas (trypansome) 35 cycles BioTechniques 24 Aug 2010: Cheap PCR: new low cost machines challenge.. 2. Rapid Diagnosis rrt- PCR on-site diagnosis for animal pathogens/. E.g. Portable PCR cycler machines are used to test cattle for TB Detection of Biological Agents Used for Terrorism: Are We Ready...by M Kellogg Nov as a portable PCR machine that reports a quantitative value.... after the First Gulf War because the rapid tests used in that war were Similar After 35 cycle there will theoretically be 2 36 = 68 billion copies of the target sequence 1 billion bases ~ 1 picogram (10-12 ) 68 billion of 2000 base fragment = 1.4 micrograms if starting from one template molecule. 7

8 ! Major application: genotyping of alleles based on a Restriction Fragment Length Polymorphism (RFLP) Design forward and reverse primers for the gene of interest! Genotyping based on allele-specific length differences Amplification of alleles that show length polymorphisms: Variable Number Tandem Repeats (VNTRs) Microsatellite sequences are one type of VNTR e.g. (CA)n Widely dispersed throughout the human genome PCR product Allele A (CA)3 Allele A contains an EcoRI restriction site EcoRI GAATTC GGATTC Allele B lacks the EcoRI site due to a single base mutation Allele B (CA)8 The alleles differ by a Single Nucleotide Polymorphism (SNP) Gel electrophoresis of PCR products digested with EcoR1 from different individuals AA BB AB Homozygotes: AA, BB Heterozygote: AB Agarose gel showing PCR products AA AB BB Homozygotes: AA, BB Heterozygote: AB! PCR can be used to amplify several VNTR loci simultaneously Multiplex genotyping using fluorescently labelled primers designed to amplify alleles of different sizes: these can be separated on the same gel Alec Jefferies (1985) M M M VNTR Locus 1 VNTR Locus 2 VNTR Locus 3 Multiplexing allows alleles at many different loci to be investigated simultaneously Major applications in 1) Forensic science 2) Paternity testing 3) Population Genetics 4) Evolutionary genetics Colin Pitchfork: First person convicted using DNA Forensics. Norborough Village Murder ( Lynda Mann, 1983, Dawn Ashworth, 1986) Stephen-Lawrence-trial-Gary- Dobson-David-Norris-guilty-murder.html 8