Genetic Research “Big Picture” of Genetic Research Restriction Enzymes Properties Discovery Use in genetic research Polymerase Chain Reactions (PCR) Principles Use as modern tool in genetic research Visualization of DNA using Agarose Gel Electrophoresis

Genomic Studies Complex pattern of gene expression Sequencing of differential on/off of large number of genes Sequencing of Human Genome (2003, 13yrs) 30,000-40 000 genes 3.1billion bp Post tranaslational mechanisms also contribute to this diversity and complexity

Genomic Studies Complex pattern of gene expression Sequencing of differential on/off of large number of genes Sequencing of Human Genome (2003, 13yrs) 30,000-40 000 genes 3.1billion bp Drosophila (14 000) Nematode (20 000) Post tranaslational mechanisms also contribute to this diversity and complexity

Genomic Studies and/or chemical modification Alternative splicing modulating gene expression Post-transcriptional mechanisms Complex pattern of gene expression differential on/off of large number of genes and/or chemical modification Sequencing of Human Genome (2003, 13yrs) 30,000-40 000 genes 3.1billion bp Proteomic Studies 500,000-1 million proteins 40-60 % of genes alternatively spliced 1 gene = Avg of 3-4 isoforms 5% of genes alternatively spliced Drosophila (14 000) Nematode (20 000) Post tranaslational mechanisms also contribute to this diversity and complexity

Genomic Studies Alternative splicing modulating gene expression Post-transcriptional mechanisms Complex pattern of gene expression differential on/off of large number of genes and/or chemical modification Sequencing of Human Genome (2003, 13yrs) 30,000-40 000 genes 3.1billion bp Proteomic Studies 300,000-1 million proteins 40-60 % of genes alternatively spliced 1 gene = Avg of 3-4 isoforms 5% of genes alternatively spliced Drosophila (14 000) Nematode (20 000) Purpose is to define meaningful knowledge of genome sequence Identify When, Where, Why and How genes are expressed 50-60% of discovered human genes have unknown functions Post tranaslational mechanisms also contribute to this diversity and complexity

Genomic Studies Alternative splicing modulating gene expression Post-transcriptional mechanisms Complex pattern of gene expression differential on/off of large number of genes and/or chemical modification Sequencing of Human Genome (2003, 13yrs) 30,000-40 000 genes 3.1billion bp Proteomic Studies 300,000-1 million proteins 40-60 % of genes alternatively spliced 1 gene = Avg of 3-4 isoforms 5% of genes alternatively spliced Drosophila (14 000) Nematode (20 000) Purpose is to define meaningful knowledge of genome sequence Identify When, Where, Why and How genes are expressed 50-60% of discovered human genes have unknown functions FUNCTIONAL GENOMICS Post tranaslational mechanisms also contribute to this diversity and complexity Transcriptomics Proteomics Structural genomics Comparative genomics

Genomic Studies How proteins interact Alternative splicing modulating gene expression Post-transcriptional mechanisms Complex pattern of gene expression differential on/off of large number of genes and/or chemical modification Sequencing of Human Genome (2003, 13yrs) 30,000-40 000 genes 3.1billion bp Proteomic Studies 300,000-1 million proteins 40-60 % of genes alternatively spliced 1 gene = Avg of 3-4 isoforms 5% of genes alternatively spliced Drosophila (14 000) Nematode (20 000) Purpose is to define meaningful knowledge of genome sequence Identify When, Where, Why and How genes are expressed 50-60% of discovered human genes have unknown functions FUNCTIONAL GENOMICS Post tranaslational mechanisms also contribute to this diversity and complexity How proteins interact with each other and the environment Transcriptomics Proteomics Structural genomics Comparative genomics Normal Abnormal

Genome-wide assessment of mRNA transcripts Microarray Studies Genome-wide assessment of mRNA transcripts Whole process is based on hybridization of probe to target DNA GREEN: Control DNA, RED:Experimental DNA Microarrays contain up to 30,000 target spots Microarray type Application Comparative Genomic Hybridization Tumor classification, risk assessment, and prognosis prediction Expression analysis (transcriptomics) Drug development, drug response, and therapy development Mutation/Polymorphism analysis Drug development, therapy development, and tracking disease progression

SIGNALING NETWORK FOR EGFR FAMILY 1 2 3 4 X X X P- -P -P Shc Grb2 p85 GAP Gab1 SHP-1 Sos p110 Src Grb7 PLC-γ Crk STAT Cbl Raf Eps Ras Dok-R MEK Akt Nck PKC Ca++ Erk CaMK JNK FOX Sp1 c-jun c-myc NF-κB c-fos Elk Ets Stat Transcription PROLIFERATION ANGIOGENESIS DIFFERENTIATION SURVIVAL MIGRATION ADHESION Transgenic Knockouts/Knockins Condidional Expression Adapted f rom Holbro et al. Ann Rev Pharmacol Toxicol., 2004, p195.

TESTING THERAPY IN ANIMALS Shc Grb2 p85 GAP Gab1 SHP-1 Sos p110 Src Grb7 PLC-γ Crk STAT Cbl Raf Eps Ras Dok-R MEK Akt Nck PKC Ca++ Erk CaMK JNK FOX Sp1 c-jun c-myc NF-κB TCR c-fos Elk Ets Stat

TESTING THERAPY IN ANIMALS BASIC SCIENCE CLINICAL TRIALS INTERVENTION THERAPY TESTING THERAPY IN ANIMALS OUTCOME ELUCIDATION OF MECHANISMS PATIENT SAMPLES SURVIVAL Ras MEK Akt Nck PKC Ca++ Dok-R HUMAN CELL LINES Erk CaMK JNK BILOGICAL ASSOCIATIONS FOX Sp1 c-jun c-myc NF-κB c-fos Elk Ets TCR Stat

Early 1970s: Genomic Studies: How to cut DNA into manageable fragments? Chemical/mechanical means non-specific, non-reproducible Breakthrough needed Restriction Endonucleases: Molecular Scissors for Cutting DNA Recognition sequence (6 bp) Binding of endonuclease to recognition site Cutting of DNA producing “overhanging”/“sticky”ends (Can also produce blunt ends)

Restriction enzymes are usually homodimers

Restriction Enzymes Recognize Palindromic Sequences ?

Restriction Enzymes Recognize Palindromic Sequences (Inverted Repeats) DNA Sequence Recognized EcoRI 5'GAATTC 3'CTTAAG BamHI 5'GGATCC 3'CCTAGG                               HindIII 5'AAGCTT 3'TTCGAA MstII 5'CCTNAGG 3'GGANTCC                                 TaqI 5'TCGA 3'AGCT NotI 5'GCGGCCGC 3'CGCCGGCG AluI 5'AGCT 3'TCGA

Restriction enzymes can be used to create a “MAP” of DNA Cleavage of DNA with restriction enzymes provides landmarks and sequence information. ? MAP ?

Restriction enzymes can be used to create a “MAP” of DNA Restriction enzymes played important role in cloning of the Human Genome Digestion of DNA with restriction Enzymes, cloning into vectors Construction of restriction maps of individual clones Overlapping clones identified by use of computers Overlapping clones arranged into contigs

FORMATION OF RECOMBINANT DNA

How were restriction enzymes discovered?

DISCOVERY FROM BACTERIAL CELLS

How is bacterial DNA protected from restriction enzyme digestion?

Bacterial methylase adds methyl group to one or 2 base pairs leading to a restriction-modification system Example: In some bacteria, cytosine nucleotide has an extra single carbon group added to it. Restriction endonucleases no longer recognize the methylated DNA and hence are protected from digestion. This is how restriction enzymes got their names: They restrict the synthesis of foreign DNA

Organism from which derived Target sequence (cut at *) 5' -->3' Restriction enzymes recognize palindrome sequences Enzyme Organism from which derived Target sequence (cut at *) 5' -->3' Ava I Anabaena variabilis C* C/T C G A/G G Bam HI Bacillus amyloliquefaciens G* G A T C C Bgl II Bacillus globigii A* G A T C T Eco RI Escherichia coli RY 13 G* A A T T C Eco RII Escherichia coli R245 * C C A/T G G Hae III Haemophilus aegyptius G G * C C Hha I Haemophilus haemolyticus G C G * C Hind III Haemophilus inflenzae Rd A* A G C T T Hpa I Haemophilus parainflenzae G T T * A A C Kpn I Klebsiella pneumoniae G G T A C * C Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii C T G C A * G Sma I Serratia marcescens C C C * G G G SstI Streptomyces stanford G A G C T * C Sal I Streptomyces albus G G * T C G A C Taq I Thermophilus aquaticus T * C G A Xma I Xanthamonas malvacearum C * C C G G

Polymerase Chain Reaction ?

POLYMERASE CHAIN REACTION STEPS INVOLVED Reverse Primer Forward Primer Taq Polymerase

Unlike most enzymes, Taq polymerase can withstand high temperatures necessary for DNA strand separation and can be left in the reaction.

PCR: Representative Temperature Profile

POLYMERASE CHAIN REACTION AMPLIFICATION OF DNA

POLYMERASE CHAIN REACTION (Con’t)

EXPONENTIAL AMPLIFICATION OF PRODUCT

What reagents are missing? STEPS INVOLVED Taq Polymerase Forward Primer Reverse Primer  template DNA  primer 1  primer 2  dNTP  Taq polymerase ?

Role of MgCl2 in PCR What could happen if [MgCl2] is too low or too high?

DESIGNING PRIMERS 1.  Primers should be 18-25 bases in length;   2.  Base composition should be 50-60% (G+C);   3.  Primers should end (3') in CG or GC: this creates “tight” ends and increases efficiency of priming   4.  Tm = 4(G + C) + 2(A + T) oC: Ta should be 2-5 oC below Tm( 55-80oC) 5.  Primers should not be complementary: primer-dimer 6.  Primer self-complementary (ability to form hairpins) should be avoided.  Adapted from Innis and Gelfand, 1991

AGAROSE GEL ELECTROPHORESIS Agarose: A polysaccharide extracted from seaweed. Used to separate DNA fragments based on size

Detecting DNA using Ethidium Bromide Structure of Ethidium Bromide (Fluorescent Dye) Intercalates between bases of DNA When excited by UV light, EtBr emits fluorescent light at 590 nm

How are different sizes of DNA strands separated on agarose gel? Mixture of DNA molecules

DNA separation is based on fragment size Decreasing Size (+) (-) PCR1 PCR2

Varying concentrations of agarose DNA Ladders How does one make a 0.7%, 1.0% & 1.5% agarose gel? Why are there varying concentrations of agarose gels?

Varying concentrations of agarose DNA Ladders How does one make a 0.7%, 1.0% & 1.5% agarose gel? Why are there varying concentrations of agarose gels? (Depends on length of fragment to be separated) Higher concentrations provide better resolution for smaller DNA fragments Lower concentrations provide better resolution for larger DNA fragments

TIPS ON HOW TO DO WELL IN “WARM-UP” LAB

of microcentrifuge tube The major reason for this experiment not to work is not having all the reaction components in the correct tube. Place droplets on wall of microcentrifuge tube Tick list

 Component  Reaction 1  Reaction 2  template DNA  1ml  primer 1  primer 2  -  dNTP  2ml  10X taq buffer  5ml  50mM MgCl2  3ml  water  37.8ml  36.8ml  Taq DNA polymerase  0.2ml  Total volume  50ml Make Master Mix containing dNTPs, Buffer, MgCl2: To save time, you can calculate how much of each to add before coming to the lab.

Tick list for PCR Reactions  Component  Reaction 1  Reaction 2  template DNA  1ml √  1ml  primer 1  primer 2  -  dNTP  2ml  10X taq buffer  5ml  50mM MgCl2  3ml  water  37.8ml  36.8ml  Taq DNA polymerase  0.2ml  Total volume  50ml

Tick list for DNA Digest    Tube 1 Tube 2 Tube 3  Tube 4 Tube 5  DNA  1ml √  1ml  10X buffer  2ml √ 2ml √  2ml  EcoRI  0.5ml √  0.5ml  BamH1  HindIII  water  16.5ml  16ml  total  20ml

Droplet method Check pipet tip for solution. Tap eppendorf Check pipet tip for solution. Eject solution on the side of tube. Check for a droplet To get the solution in the droplet to the bottom of the tube tap the tube on the bench.

Please Balance the Microcentrifuge

Please Balance the Microcentrifuge WHY? Non-Scientific Explanation Scientific Explanation The Microcentrifuge will break! ?

Please Balance the Microcentrifuge WHY? Non-Scientific Explanation Scientific Explanation Centripetal Force (CPF) exerted by sample towards the centre of microcentrifuge. Newtons 3rd law : Every action has equal & opposite Rxn (For every CPF there Should be equal & opposite CFF) The Microcentrifuge will break Centripetal Force that the sample exerts on the rotor depends on both mass and radius.

THE END