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Basic Molecular Methods “A historical look genetic analysis” Rosalind Franklin (Image: wikipedia.com) Adam Kisailus, Ph.D. Molecular Methods What will be presented: experimental methods used for studying the molecular biology of cancer Goal of this class: Obtain an appreciation of practical application of molecular techniques Molecular analysis of BRCA Hereditary breast cancer versus sporadic breast cancer Germ line mutations to BRCA gene correlated with breast cancer susceptibility Gene was partially mapped in 1992 Gene was “discovered” in 1996 Characterized as a tumor suppressor gene which plays a role in cell cycle inhibition (1997Present) 2002- Epigenetically regulated Anatomy of a gene Gene is a coding unit for a protein Exon1 Intron Exon 2 Exon: encodes protein Intron: does not encode protein The central dogma Exon1 replication Exon 2 transcription Exon1 DNA (gene) reverse- transcription Exon 2 mRNA translation protein The central dogma in cancer cells Mutation Exon1 replication Exon 2 DNA (oncogene) transcription Exon1 aberrant mRNA translation Tumor suppressor -orTumor promoter Identification of the BRCA mutation Mutation of BRCA correlates with disease onset JM Hall et al Am. J Hum. Genet. 1992 June; 50(6):1235-1242 BRCA is defined as a tumor suppressor Casey et al Hum Mol Genet. 1993 Nov;2(11):1921-7 The central dogma in cancer cells Mutation Exon1 replication Exon 2 DNA (oncogene) transcription Exon1 aberrant mRNA Translation Tumor suppressor -orTumor promoter 1992-1996: Mapping the BRCA gene and mutation identification RFLP analysis Southern blotting PCR Kary Mullis- (Google images) Properties of DNA Complementary base pairing Anneals/denatures under varying temperature conditions, Negatively charged phosphate backbone Semi-conservative replication Source: Google Images Molecular biologists aren’t inventors, they’re opportunists How can we harness these properties of DNA to study our gene? Restriction Fragment Length Polymorphisms (RFLP) analysis Single nucleotide polymorphisms (SNPs) Used as genetic “fingerprint” Polymorphisms can be mutations in oncogenes Detection of polymorphisms by 2 component method: (1) restriction enzyme digest and (2) Southern blot analysis Restriction Enzymes Restriction Digest Enzymes Bacterial origin, defense mechanism for bacteria to fragment invading phage (bacterial virus) Recognize and cleave specific palindromic site on DNA Very straight forward usage, practically no problems except for STAR activity. Resulting digested DNA is a restriction fragment Eco RI: 5’-ATGCGC G-A-A-T-T-C GGCCT-3’ 3’-TACGCG C-T-T-A-A-G CCGGA-5’ Restriction fragments and sticky ends Eco RI: 5’-ATGCGC G-A-A-T-T-C GGCCT-3’ 3’-TACGCG C-T-T-A-A-G CCGGA-5’ Eco RI: 5’-ATGCGC G-3’ 3’-TACGCG C-T-T-A-A-5’ Can pair with Corresponding DNA bases Single Nucleotide Polymorphism (SNP) Restriction enzyme no longer recognizes cut sequence. And does not cut. Eco RI: 5’-ATGCGC G-A-G-T-T-C GGCCT-3’ 3’-TACGCG C-T-C-A-A-G CCGGA-5’ RFLP Analysis: Genetic diagnostic RFLP Analysis of Offspring’s DNA: 1. Cut from human chromosome with reference R.E. sites 2. Digested with mut BRCA (Paternal) BRCA (Maternal) specific R.E.s Restriction digest mut 2. Probe with Radio labeled ssDNA probe BRCA (Paternal) BRCA (Maternal) Gel electrophoresis Gel electrophoresis Source: Google images Southern blotting Method for analyzing structure of DNA and identifying mutations within regions of small sequence repeats Obtain genomic DNA samples from patient and both parents. Digest DNA with restriction enzymes to map mutations Probe for DNA fragments to visualize genetic profile Southern Blot analysis of BRCA gene RFLP Analysis of Offspring’s DNA: BRCA mut BRCA Paternal (P) Maternal (M) Cut sites Restriction digest BRCA mut BRCA “DNA probing” (Source:Google Images) M P M O P O Paternal (P) Maternal (M) Denaturation to ssDNA and blotting to membrane Gel electrophoresis autoradiography 1997: BRCA gene product analysis Study of BRCA 1992-1996 Know it correlates with cancer Loss of function results in tumorigenesis What is its molecular function? In vitro studies Clone the BRCA gene for analysis of protein product “Characterization of cancer-linked BRCA1-BRCT missense variants and their interaction with phosphoprotein targets.” Drikos I, Nounesis G, Vorgias CE. Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15701 Athens, Hellas. The central dogma Exon1 replication Exon 2 transcription Exon1 DNA (gene) reverse- transcription Exon 2 mRNA translation protein Molecular cloning and the birth of biotech 1980- Genentech Herb Boyer and Robert Swanson: “Science meets business- in a bar.” Recombinant insulin1978 Human Growth hormone -1985 1988- Canseco and Mcgwire the bash bro. Source: Frederic Larson Steps of molecular cloning Prepare cDNA (using RT-PCR) Prepare plasmid DNA Insert cDNA into plasmid DNA Transform bacteria and select for plasmidcontaining bacteria Confirm cDNA insertion Polymerase Chain Reaction (P.C.R.) techniques PCR: gene product amplification starting from template DNA generate DNA for cloning RT-PCR: gene product amplification starting from template RNA (recall the central dogma) Generate cDNA for cloning measure gene expression quantitative RT-PCR Screen mRNA for mutations Polymerase Chain Reaction (PCR) Supposedly developed by eccentric scientist Carey Mullis while he was driving the west coast highway “tripping on acid” Components DNA polymerase originating from Thermos Aquaticus (Taq polymerase) to withstand high temperatures Polymerases vary by fidelity and range depending on usage deoxynucleotides (dCTP, dATP, dGTP, dTTP) in equal amounts! Oligo primers: designed to specified gene, length and GC content important for annealing Co-enzymes: MgCl and buffer Properties of DNA Complementary base pairing Anneals/denatures under varying temperature conditions, depends on sequence Negatively charged phosphate backbone Semi-conservative replication P.C.R. Thermocycling 27-33 cycles + 5’ 3’ - 3’ 5’ 5’ 3’ 3’ 5’ Taq 5’ 3’ Taq 3’ 5’ 94o “DNA template strand separation” 53o “Primer annealing” 72o “Primer extension” RT-P.C.R.: Making cDNA for Cloning Trizol® extraction of mRNA from normal tissue: mRNA is solubilized in aqueous phase while organic phases precipitate lipids/protein/DNA 1.) First strand synthesis: Primer: Oligo (dT) +reverse transcriptase mRNA 5’ DNA 3’ 2.) Digestion of RNA By RNaseH mRNA 5’ AAAA-OH 3’ TTTT 5’ mRNA 5’ AAAA-OH 3’ TTTT 5’ 3.) Use mRNA fragments As primers for DNA pol I (klenow fragment) for second strand synthesis 3.) Second strand synthesis Completion Pol I. “5’ “3’ 3’ ” AAAA-OH 3’ TTTT 5’ 5’ ” AAAA-OH 3’ TTTT 5’ cDNA 5’ 3’ (Product DNA) Visualizing P.C.R. Product Gel electrophoresis Ethidium Bromide (Source: Google images) Plasmid DNA Plasmid DNA is used for cDNA expression in prokaryotic/eukaryotic cells Anti-biotic resistance selection marker (1) Origin of replication (2) Multiple cloning site (3) Protein fusion tag (4) (4) (3) (2) (1) “Plasmid DNA Map” (Source: NEB catalogue) Putting it all together: Cloning of BRCA cDNA schematic A. BRCA cDNA B ATG PCR using Oligos incorporating restriction enzyme sites R ATG MCS E X Linearize vector by E digestion X D. C. Transformation of bacteria R X ligation Selection on antibiotic E Propagation in culture (cloning) Confirm BRCA gene is inserted in vector PCR check Digestion diagnostic Sequencing- “Old School” Method DeoxyHH (-OH available Di-Deoxyto make phosphate Back bone) GGGGTTTAAG 4 Reactions- contains 4 deoxy-nucleotides And 1 dideoxy-nucleotide of either A,C,T,G dATP dTTP dGTP dCTP ddXTP-PCR stopper, because of dideoxy nature There is no available –OH group for adding another base. P32 labeled and will appear as band on autoradiograph Polymer chain + ddG ddA ddT ddC PCR products Run on gel - Confirm BRCA gene is inserted in vector “New school” method of PCR Take primer and plasmid to core facility Automatic fluorescent sequencing: Incorporation of dye-labeled ddNTPs into DNA extension products. Each dye emits a different wavelength when excited by argon ion laser so that all four bases are detectable in a single lane of the gel. GGGGTTTAAG PCR products Run on gel ddATP ddTTP ddGTP ddCTP Things to consider when cloning a gene Restriction enzyme usage Use an internet software tool like NEB®Cutter to ensure the restriction sites you select occur nowhere else within the gene Use two different restriction enzymes for directionality of insertion Always, always, when available use enzymes which leave sticky ends, avoid blunt-end ligation http://tools.neb.com/NEBcutter2/index.php Things to consider when cloning a gene (cont.) Oligo design Include a GAG sequence to ensure that the restriction enzyme has enough DNA “scaffold” Make sure that no more than 3 bases of forward and reverse oligos are complimentary to avoid “primer dimers” http:// www.basic.northwestern.edu/biotools/oligocalc.html Temperature of melting (50-55 degrees C) depends on GC content and length of oligos Forward primer, use – strand as template: 5’-GAGGAATTCATGCCGTACGGCCGG-3’ Reverse primer, use + strand as template 5’-GAGCTCGAGCTACTTCGGGGCCCAG-3’ 15 bases into coding region + 5’ - 3’ 3’ http://www.basic.northwestern.edu/biotools/oligocalc.html http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=27886591 Things to consider when cloning a gene (cont.) Usage of shuttle vector Taq polymerase leaves behind a 5’ T and 3’ A overhang TOPO shuttle vector has these overhangs and therefore your PCR product with restriction sites intact can be easily inserted into the TOPO shuttle vector, propagated and then cut with restriction enzymes Cutting PCR product with restriction enzymes not very efficient Ligation conditions Linearized vector must be gel purified Use 5 to 10 fold excess amount of insert to vector to drive ligation reaction Bacterial Transformation Transformation: Inserting plasmid DNA into bacteria DH5-alpha: Suitable cell line for cloning Steps involved in transformation: 1.) Inoculate competent cells with plasmid DNA 2.) Heat shock 3.) Recover in LB media without antibiotic 4.) Select for bacteria which uptook plasmid DNA on media containing antibiotic for selection Confirming BRCA gene is inserted in vector Extract DNA from bacteria Buffer steps: 1.) Resuspend overnight culture of bacteria in resuspension buffer and RNase 2.) Lyse bacteria in SDSa detergent and NaOH a base 3.) Neutralize in acidic buffer of sodium acetate 4.) Precipitate DNA in 2propanol 5.) Wash in 70% ethanol 6.)Resuspend pellet in TE buffer 1.) 2.) 3.) http://learn.genetics.utah.edu/units/biotech/ Measuring gene expression levels RT-PCR mRNA Northern blot analysis mRNA Western blot analysis protein Genes Chromosomes Cancer. 1997 Sep;20(1):53-9. “Analysis of the BRCA1 and BRCA2 genes in sporadic meningiomas.” Kirsch M, Zhu JJ, Black PM. Utilize BRCA cDNA to analyze BRCA gene expression and structure Southern blot – gene mutation replication and structure analysis “DNA probe for genomic DNA” Northern blot- gene expression “DNA probe for RNA” Western blot- protein expression ”antibody probe for protein detection” Far Western blot- protein interaction ”protein overlay for protein:protein interaction” Exon1 Exon 2 DNA (gene) transcription Exon1 Exon 2 mRNA translation protein Quantitating BRCA gene expression by real time-PCR (not RT-PCR) Quantitation of low amounts of mRNA or low copy number DNA using PCR relies on the concept that DNA binding dyes (Envision®,Sybr green) fluoresce when incorporated into double stranded DNA) Eliminates the need for post-PCR processing or gels. + 5’ 3’ 94o “DNA template strand separation” - 3’ 5’ 5’ 3’ 3’ 5’ 5’ 27-33 cycles Taq 3’ 53o “Primer annealing” 72o “Primer extension” Digital measurement of signal. Northern Blot analysis of BRCA gene expression in cancer cell line N 1o M N Transfer mRNA membrane Gel electrophoresis 1o M N 1o M Expose to film Probe with P32-labeled BRCA cDNA Note: in silico northerns for most genes are available at NCBI. Auto-radiograph Protein Analysis: Exploiting properties of proteins Proteins are composed of -/+ charged, inert, acidic and basic amino acids Amino acid sequences are antigenic Proteins are heat and organic chemicaldenatured (Source: Google images) Protein Electrophoresis 1. - - - 2. - - 1 2 SDS binds to amino acid residues and gives uniform negative charge to protein; heat/mercaptoethanol denatures/linearizes protein Protein 3. 1 2 - - Add protein sample to SDS-PAGE Gel lane 2. Electric current Protein separated by “APPARENT” molecular weight Adapted from molecularstation.com Western Blot Analysis: Immunodetection Immunodetection of protein in cell lysate. Components Primary antibody: has affinity for target protein Monoclonal: Recognizes specific epitope on protein, made in mouse Polyclonal: Antibody made to whole protein typically in rabbit Secondary antibody: has affinity for primary antibody Typically generated in goat Cost-effective Signal Secondary antibody is conjugated to a signal producing enzyme or fluor • Alexa Fluor: Emits light when excited by infrared beam (LiCor) • Horse Radish Peroxidase: Chemical substrates added which are metabolized by HRP generating chemiluminescent signal. Western Blot Analysis:Blotting 2o antibody Cell lysates N Ca - 1o antibody + Block non-specific Sites on membrane (Antibody detection) N Ca Denaturation of protein And uniform neg. charge. Transfer to membrane SDS PAGE gel separation by charge. Signal detection on film Method for studying gene function controversy: Knocking out a gene versus overexpressing a gene Overexpression of some genes such as those which encode scaffolding molecules may result in artifact; however, in other cases it can provide function of a protein’s activity Transient protein expression Stable protein expression Silencing or knocking down gene expression gives clearer indication of gene function RNA interference (RNAi) Transfection of mammalian cells Vector Mammalian expression vectors Bacterial expression vectors Delivery Method DEAE-dextran- old school method; precipitation between DEAE (+charge) and DNA, uptake by pinocytosis-? Lipofection- use of liposome/DNA complex (Fugene®; Lipofectamine®) Electroporation- use of electric current, efficient means, but kills almost 90% of the cells. Comparison of mammalian expression and bacterial expression vectors MCS TAG MCS Antibiotic selection Stable Transf. Bacterial expression vector: Mammalian expression vector (Source: NEB Catalogue) 2001: Understanding the function of BRCA by expression “knockdown” Nature Paper 1998- Andrew Fire and Craig Mello, 2006 Nobe Laureates Antisense inhibition of BRCA1 expression and molecular analysis of hereditary tumors indicate that functional inactivation of the p53 DNA damage response pathway is required for BRCA-associated tumorigenesis. Reedy MB, Hang T, Gallion H, Arnold S, Smith SA. RNAi and siRNA:Andrew Fire and Craig Mello 1998 siRNA is complementary to mRNA, forming a double stranded complex resulting in its degradation and inhibiting translation into protein Exon1 Exon 2 FAK Gene (DNA) mRNA siRNA NH protein COOH ? signaling “Central dogma” degraded siRNA knockdown of BRCA Introduce siRNA for BRCA1 into cancer cell Knock down BRCA1 levels, treat with chemotherapy to determine drug resistance affects Oncogene. 2009 Jan 29;28(4):575-86. Epub 2008 Nov 10 “Decreased BRCA1 confers tamoxifen resistance in breast cancer cells by altering estrogen receptor-coregulator interactions.” Wen J, Li R, Lu Y, Shupnik MA. 2002-2009: BRCA characterization and function Domain mapping Protein:Protein interactions Enzymatic activity “Characterization of cancer-linked BRCA1-BRCT missense variants and their interaction with phosphoprotein targets.” Drikos I, Nounesis G, Vorgias CE. Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15701 Athens, Hellas. Characterization of signaling domains by site-directedmutagenesis Following wholesale knockout of gene expression we’d like to carefully map a binding site or activity Site-directed-mutagenesis enables precise alterations to amino acids by relying on design of a pair of oligo primers with mismatched base pairs. Site-directed-mutagenesis CH3 Mammalian Expression vector CH3 MOVE Not I digestion Leu Gly Ala Asn Leu TTA GGA GCT AAT TTA AAT CCT CGA GCA AAT Leu Gly Ala Ala Leu ATP binding domain Pfu polymerase Extension. Transfection And expression Pfu polymerase Extension, nick synthesis. Which amino acid to substitute? Alanine, which has a small inert side chain is typically used for aa substitution in Site-directed-mutagenesis. However, one must take into account structure in Cases of amino acids so as not to disturb secondary structure of the protein and Most closely mimic the original amino acid which has been substituted. END