© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Chapter 2 Technical Foundations of Genomics Recombinant-DNA techniques used in genomics

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Contents  Introduction  Genomic and cDNA libraries  Hybridization and Northern blots  Subcloning  Restriction-enzyme mapping  DNA sequencing  PCR amplification  Protein expression

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Introduction  Genomics built on recombinant-DNA technology  Thorough understanding of recombinant-DNA techniques  Prerequisite for understanding genomics technologies  Differences between genomics and recombinant-DNA technology  Genomics is high throughput  Genomics is dependent on computational analysis

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Genomic and cDNA libraries  Libraries are fragments of DNA cloned into a vector  Libraries are usually constructed before sequencing  Genomic libraries are used for genomewide sequencing  cDNA libraries are needed for EST sequencing

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Genomic library  Made from fragments of genomic DNA  Genomic DNA cut up with restriction enzymes or randomly broken by mechanical shearing  Fragments ligated into cloning vectors  Small insert  Lambda phage  Plasmid  Large insert  BACs

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey How to make a genomic library total genomic DNA plasmid (black) amp R ori amp R ori amp R restriction enzyme same restriction enzyme anneal and ligate transform E. coli; select for Amp resistance genomic DNA

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Making a cDNA library  Step 1: Isolate RNA  RNA is purified from tissue or cell line  The mRNA is then isolated away from ribosomal and tRNAs  Column with oligo dT is used to bind poly A stationary support polyT mRNA polyA tissue or cell

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Step 2: Obtain cDNA from RNA  mRNA is treated with the enzyme reverse transcriptase  The enzyme copies sequence of mRNA into first strand of DNA  Another enzyme is used to make second strand of cDNA

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Step 3: Transformation  Double-stranded cDNA is inserted into cloning vector  cDNA is ligated into cloning vector (plasmid or phage)  Vector is transformed or infected into bacteria E. Coli bacteria plasmid

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Step 4: Library screening  Colony DNA is attached to membrane  DNA is screened with labeled probes  DNA is labeled with radioactivity  Labeled DNA is allowed to hybridize with DNA on membrane  After washing, positive hybridization spots are identified Radioactive probe selected colonies membrane hybridization X-ray film

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey cDNA to EST  For use in EST sequencing  Need to array individual clones  Library is spread on bacterial plates  Individual colonies are picked  Colonies are placed in test tubes or microtiter plates cDNA library Clone 12345

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Colony picking  Automatic colony pickers play key role in genomics  Instead of manually picking one colony at a time, they identify and pick multiple colonies from plates  Pickers then deposit each colony into a microtiter well

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Hybridization  Basis of microarrays for determining gene expression  Process by which complementary strands find each other  A–T and C–G base pairing  Dependent on temperature, salt, sequence, and concentration

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Northern blot  Gene expression analyzed by Northern blots  RNA samples undergo electrophoresis  RNA separated by molecular weight  Transferred to membrane  Probe labeled  Radioactivity or antibody ligand  Hybridized to RNA on membrane  Hybridization dependent on time, temperature, salt concentration, and nucleic acid sequence and concentration

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Steps in Northern blotting

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Northern blot example  Example of time course of gene induction  Upper panel: RNA after electrophoresis  Bands correspond to ribosomal RNA  Probe detects two bands  Lower panel: Lower band shows rapid induction and then decline  Upper band shows slower induction, but stays induced for longer Time after elicitation – 4.2 – 2.1

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Northern blot and microarray hrs hrs DMC1 – SPS1 – DIT1 – SPS100 – hrs DMC1 – SPS1 – DIT1 – SPS100 – fold repressed fold induced >20 10 x 3 x | 3 x 10 x >20 1:1

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Cross-hybridization  Hybridization to a related, but not identical, sequence = cross-hybridization  Example: A probe from one member of a gene family is likely to hybridize to all other members  Problem in microarrays, particularly cDNA arrays  Oligonucleotide arrays prescreened to eliminate sequences likely to cross-hybridize

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Subcloning  Propagating fragments of cloned DNA  Used for sequencing and protein production  Plasmid vectors  Replicate in bacteria  Resistant to antibiotics  Cloning sites Region into which DNA can be inserted Plasmid cloning vector ORI amp r

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Subcloning: vector and fragment  Vector and fragment to be inserted must have compatible ends  Sticky ends anneal  Enzyme ligase makes covalent bond between vector and fragment  Use of recombination instead of restriction sites restriction enzymes DNA cloning vector fragment recombinant plasmid

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Recombination cloning  Uses site-specific recombination for subcloning  DNA fragment flanked by recombination sites  Add recombinase “Clonase ® ”  Moves fragment from one vector to another

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Transformation into bacteria  Bacteria prepared for transformation by making outer membrane permeable to DNA  Become competent  DNA added to bacteria  Heat shock  Plate on selective media E. coli host cell transformed cell recombinant plasmid

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Restriction-enzyme mapping  Used for physical mapping of DNA  Restriction enzymes cut at defined sites  Palindromic sequences  Sites are landmarks on DNA  Then fragments are separated by gel electrophoresis CGATCG GCTAGC

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Gel electrophoresis  DNA fragments are separated by size in electric field  DNA negatively charged: proportional to size of fragment  Separated through gel matrix  Agarose or acrylamide

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Cutting a BAC with restriction enzymes  Separate DNA fragments are cut with restriction enzyme  DNA is visualized with ethidium bromide  Binds to DNA and fluoresces orange  The sizes of the fragments are determined based on a standard Log MW Distance....

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey DNA sequencing  Most current sequencing projects use the chain termination method  Also known as Sanger sequencing, after its inventor  Based on action of DNA polymerase  Adds nucleotides to complementary strand  Requires template DNA and primer

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Chain-termination sequencing  Dideoxynucleotides stop synthesis  Chain terminators  Included in amounts so as to terminate every time the base appears in the template  Use four reactions  One for each base: A,C,G, and T 3’ ATCGGTGCATAGCTTGT 5’ 5’ TAGCCACGTATCGAACA* 3’ 5’ TAGCCACGTATCGAA* 3’ 5’ TAGCCACGTATCGA* 3’ 5’ TAGCCACGTA* 3’ 5’ TAGCCA* 3’ 5’ TA* 3’ Sequence reaction products Template

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Sequence detection  To detect products of sequencing reaction  Include labeled nucleotides  Formerly, radioactive labels were used  Now fluorescent labels  Use different fluorescent tag for each nucleotide  Can run all four reactions in same lane TAGCCACGTATCGA A* TAGCCACGTATC* TAGCCACG* TAGCCACGT*

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Sequence separation  Terminated chains need to be separated  Requires one-base-pair resolution  See difference between chains of X and X+1 base pairs  Gel electrophoresis  Very thin gel  High voltage  Works with radioactive or fluorescent labels – + C A G T

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Sequence reading of radioactively labeled reactions  Radioactive labeled reactions  Gel dried  Placed on X-ray film  Sequence read from bottom up  Each lane is a different base ATCG + –

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Sequence reading of fluorescently labeled reactions  Fluorescently labeled reactions scanned by laser as particular point is passed  Color picked up by detector  Output sent directly to computer

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Summary of chain termination sequencing

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Polymerase chain reaction  Used in sequencing, diagnostics, comparative genomics, etc.  Uses thermostable DNA polymerase  Able to function near boiling temperature  Two primers complementary to sequences at 5’ and 3’ of region to be amplified  Double-stranded DNA template  Performed in thermal cyclers programmed to raise and lower temperature

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey PCR machines

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey PCR reaction: annealing primers  Template melted into two strands by high heat  > 90 degrees C  Primers anneal to both strands  Polymerase makes a copy of both strands

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey PCR reaction: amplification  Temperature raised to melt newly made DNA  Primers allowed to anneal as temperature drops  Polymerase elongates new second strand of DNA  Process repeated  Exponential increase in DNA

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Protein expression  Important for proteomics  Need large amounts of recombinant protein for the following:  Structure determination  Antibody production  Protein arrays  Proteins made in bacteria, yeast, and insect cells  Then must purify the recombinant protein away from other proteins

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Protein expression vectors  Protein expression vectors have the following:  Inducible promoters  Tags for purification  Histidines  Epitopes  Proteins  Coding sequence inserted in frame

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Making recombinant protein  Expression vector transformed into bacteria  Bacteria grown to saturation  Compound added for induction  e.g., IPTG  Protein accumulates in bacteria

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Protein purification  Contents of bacteria run over column  Tagged proteins bind to column  Examples  Nickel column for His- tagged proteins  Anti-myc antibody column for Myc- tagged proteins  Elution yields purified protein

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Recombinant protein  Gel electrophoresis of recombinant protein shows the following:  Soluble proteins  Column flow-through  Purified protein  Four fractions from column 1 SHR::MBP SDS-PAGE kDa 124 – 83 – 42 – 24567

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Summary  Libraries  Hybridization and Northern blots  Subcloning  Restriction-enzyme mapping  Sequencing  PCR  Protein expression