Final

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Final

• Final 2 of the following 3 choices,
• 1 hour exam covering recent materials,
• 2 page review of an assigned paper (due June 11),
• Self-study of a remaining chapter in the text,
  answers to the odd problems.

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How are we different?at the DNA level.
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Southern Analysis

• DNA hybridizing to DNA,

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DNA Polymorphisms

• a DNA locus that has two or more sequence
  variations, each present at a frequency of 1 or
  more in a population,
• 1 in 700 frequency common in most species,
• less than 1 million loci in humans (1 in 3000).
• five classes of polymorphisms.

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Polymorphisms

• Single Base Pair Differences,
• Microsatellites (short sequence repeats),
• Minisatellites (long sequence repeats),
• Deletions,
• Duplications.

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Single Base Differences
5-------G-A-A-T-T-C--------3
3-------C-T-T-A-A-G--------5
5-------G-A-G-T-T-C--------3
3-------C-T-C-A-A-G--------5
point mutations, 98 of all mutations (1 in 200
bp).
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Point Mutations

• Chemical Mutagens,
• UV-light,
• other natural and synthetic causes.

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Detecting Point Mutations

• Direct DNA Sequencing,
• RFLP analysis,
• Alleles Specific Oligonucleotides...

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Allele-Specific Oligonucleotides

• short synthetic DNA probes able to differentiate
  between sequences differing by as little as 1
  base pair,

--AGTAGCTGTAGCT-- --TCATCGACATCGA--
Probe with fluorescent or radioactive
CTPs. Complementary, binding.
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Hybridization

• the process of joining two complementary strands
  of DNA together,
• ...or one DNA strand to an RNA strand,
• molecular probes are hybridized to complementary
  strands,
• probe/target complexes are made visible by
  fluorescence or radioactivity.

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ASO and PCR

• subject gene is amplified using PCR,
• bound to a solid substrate,
• probed with ASOs.

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Put DNA on filter, probe with the ASO.
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Microsattelites

• DNA element composed of 15-100 tandem repeats of
  one-, two- or three base sequences,

AAAAAAAAAAAAAAAAAAAAAAAAAAA
CACACACACACACACACACACACACACA
CATCATCATCATCATCATCATCATCATCAT
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Microsattelite Details

• ...also termed simple sequence repeats (SSRs),
• 1 in 30,000 bp,
• 100,000 microsattelites in humans,
• new alleles arise at an average rate of 10-3 per
  locus,
• higher than background mutation rate (10 -5),
• at 1/1000, doesnt occur too often.

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DNA Polymerase Slippage
replication incorporates repeats...
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Minisattelites

• DNA element composed of longer (10-80 bp) tandem
  repeating units of identical sequence,
• variable number of tandem repeats (VNTRs),
• often result in polymorphic region ranging from 1
  to 15 kb in length,
• ...approximately 1000 per human genome.

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Unequal Crossing Over
...minisattelite mutation frequency, 10-3.
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Deletions, Duplications, etc.

• single base pair up to many megabase deletions
  or duplications,
• cause by mutagens, or unequal crossing over,
• extremely rare, infrequent in the genome.

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Detecting Polymorphisms

• RFLPs Classical restriction fragment length
  polymorphisms,
• Advantage dont need sequence information,
• Disadvantage need lots of DNA and radioactive
  detection, labor and time dependent,
• Major Use genotyping in absence of sequence
  info.

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Detecting Polymorphisms

• RFLPs PCR restriction fragment length
  polymorphisms,
• Advantage dont need lots of DNA or
  radioactivity,
• Disadvantage labor, time dependent,
• Major Use genotyping.

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Detecting Polymorphisms

• ASOs allele specific oligonucleotides,
• Advantage can detect single base pair changes,
  not dependent on specific palindromic sequences,
• Disadvantage must know wild-type sequence, and
  sequence of alternative alleles,
• Major Use genotyping.

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Microsattelite Detection

• one-, two- or three- base pair repeats,
• need sequence information from both sides of the
  microsattelite to make PCR primers,
• gel electrophoresis distinguishes differences in
  DNA length as small as two base pairs.

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Minisattelite Detection

• DNA element composed of longer (10-80 bp) tandem
  repeating units of identical sequence,
• often result in polymorphic region ranging from 1
  to 15 kb in length,
• PCR now routinely amplifies templates up to 30
  kb,
• analysis of minisattelites is similar to that of
  microsattelites.

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Use of multiple loci provides a definite DNA
fingerprint.
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Micro-, Minisattelites

• Advantages
• provide detailed DNA fingerprints for genotyping,
• specifically useful for differentiating family
  members and forensic work,
• Disadvantages
• requires sequence information and sophisticated
  statistical analysis.

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RAPDs

• random amplification of polymorphic DNA
• ...the use of non-specific DNA sequences to
  describe a specific genome.

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RAPD Premise

• short oligos of any sequence will find
  complementary sequences in some part of the
  genome,
• if two of these target sequences are correctly
  oriented and close enough together for PCR, a
  fragment will be produced.

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RAPD Primers

• Short in length, usually 10-mers, randomly
  selected, for example...
• 5 - agtcacgcag - 3 occurs approximately every
  1,000,000 base pairs, so in the human genome
  should bind at gt3000 loci.

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Random Distribution
this experiment would generate three PCR
products.
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RAPDs

• Advantages
• no requirement for sequence information,
• great for genomes that are not well
  characterized,
• can sequence bands directly, gaining insight into
  polymorphic regions of uncharacterized species,
• Disadvantages
• provides information for random loci only.

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Deletions, Duplications

• rare events,
• karyotyping,
• SSCPs,
• PCR (look for product size differences),
• direct sequencing of DNA,
• etc.

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SSCPs

• ...Single-Strand Conformation Polymorphisms
• ...gel electrophoresis detection of nucleotide
  differences in single stranded DNA molecules,
• ... folding of single stranded DNA differs when
  there are base pair differences,
• single stranded DNA migration through gel is
  partially dependent on the molecules
  conformation.

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SSCPs

• Advantages
• can screen large numbers of individuals for
  mutated genes without direct sequencing,
• can detect differences between wild-type and
  mutant genes at all base pairs,
• dont need to know sequence of mutant alleles,
• Disadvantage
• 90 maximum detection rate.

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Genetic Disease Detection

• RFLPs,
• ASOs,
• other PCR protocols,
• determine genotypes that result in diseases
  directly,

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ASO Pave Way for DNA-Chips

• the next big thing, huge arrays of DNA for
  complex genotype and phenotype analysis,
• B-Chip (Before chips),
• A-Chips (After chips.

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DNA Arrays

• DNA systematically arrayed at high density,
• virtual genomes for expression studies,
• RNA hybridization to DNA for expression studies,
• comparative genomics,
• DNA hybridization to DNA,
• inter- and intra-species comparisons, etc.
• potential yet to be developed.

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Arrays
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Probes/Targets

• ...Probes are the tethered nucleic acids with
  known sequence,
• the DNA on the chip,
• ...Target is the free nucleic acid sample whose
  identity/abundance is being detected,
• the labeled nucleic acid that is washed over the
  chip.

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DNA-Probes

• cDNA arrays, DNA arrays,
• DNA Microarrays,
• oligonucleotide arrays,
• DNA chips.

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DNA Chips

• oligonucleotides systematically synthesized in
  situ at high density.

Affymetrix DNA Chip
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Allele-Specific Oligonucleotides(DNA Chips)

• allele specific oligonucleotides (ASOs)
  recognize single base pair differences in DNA
  sequences.

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Ordered Array of ASOs
...over a million ASOs and controls can be
gridded per cm2.
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Photolithography

• the process of using an optical image and a
  photosensitive substrate to produce a pattern,

• oligonucleotide synthesis can be inhibited by a
  protection group molecule,
• the protection group can be linked by a
  photosensitive bond, and thus cleaved by light.

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Targets

• ...fluorescent targets,
• genomic DNA,
• cDNA, mRNA or cRNA for expression studies,

targets are washed over the chip for
hybridization.
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cDNA Microarrays

• ...denatured, double stranded DNA (500 - 5000 bp)
  is dotted, or sprayed on a glass or nylon
  substrate,
• ...up to tens of thousands of spots per array,

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Hybridization Detection

• fluorescent images are read by an optical
  scanner, and intensities are compared using
  algorithms to differentiate artifacts.

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DNA Chip
DNA Microarray
Probe?
Oligos
Probe?
cDNAs
Target?
mRNA Transcripts Under Two Conditions.
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Screening for Genetic Disease

• Cystic fibrosis 75 of mutations are at the
  D508 deletion site,
• 8 are in three additional specific locations in
  the gene, the rest are spread across the length
  of the gene,
• Pre-Array tests yielded only an 83 chance of
  detecting a mutation.

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Cystic fibrosis Detection

• Create a DNA chip with ASOs for wild-type Cystic
  fibrosis gene,
• approximately 4.5 kb of the 250 kb gene codes for
  the structural portion of the gene,
• 225 20-mers span 4.5 kb,
• 20 mismatches per 20-mer requires 4500 ASOs, or
  grids, plus controls.

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Creating the Mask

• computer algorithms are used to design the mask,
• creation of mask is now the limiting process,
  requires months to accomplish, and about 100,000
  per mask,
• masks have limited lifetimes, each array costs
  about 100 currently.

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Cystic fibrosis Chip

• using photlithography, create a chip with ASOs
  to identify any difference from wild-type DNA,
• match results with mutations at know deleterious
  loci,
• catalog new deleterious loci.

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1 Gene of Many

• with controls, the Cystic fibrosis gene may
  require up to 20,000 grids,
• new chips can accommodate up to 1 million grids,
• can look at 50 similarly sized genes on one
  chip.

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4000 Genetic Diseases

• as genes are linked to diseases, quick,
  inexpensive tests can be performed to determine
  who carries specific mutations,
• computer analysis will provide genome profiles
  that predict a variety of traits.

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Genome Profiling

• with 1500 SNPs now, and up to thousands
  available, genetic profiles can be made,
• choose SNPs in or near genes involved in traits
  or diseases,
• compare profiles over large populations.

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How are we different?at the RNA level.
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Northern Analysis

• DNA hybridizing to DNA,

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DNA Arrays and Expression

• grid gene-specific ASOs onto the DNA chip, or
  cDNAs onto microarrays,
• probe with labeled cDNA, genes that are
  expressed at a specific time, place or under a
  specific condition will bind to the chip for
  display.

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Genes and Targets

• once the Human Genome Project is done, all of the
  genes can be gridded,
• presently, several completely sequenced genomes
  have been gridded,
• yeast,
• E. coli,
• various bacteria,
• drug identification, fundamental research, etc.,

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Applications

• Monitor expression patterns under the
  experimental conditions of your choosing to
  determine the function of the thousands genes,
• Common expression patterns can be used to
  identify genes that are members of the same
  pathway,
• Explore expression of candidate/unknown genes.

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Gene/Drug Discovery

• genes involved in cancer and other diseases have
  been identified through a variety of techniques,
• genome expression analysis provides a means of
  discovering other genes that are concomitantly
  expressed.

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Applications

• Can study the role of more than 1700 cancer
  related genes in association with the (rest) of
  the genome,
• Define interactions and describe pathways,
• Measure drug response,
• Build databases for use in molecular tumor
  classifications,
• benign vs. cancerous, slow vs. aggressive

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Extended Applications

• Water quality testing (4 hours vs. 4 days),
• Environmental watchdogs,
• Fundamental research on non-human subjects,
• Direct sequencing of related species for
  evolutionary studies,
• etc.

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Friday

• Intra- and Interspecies Variation in Primate Gene
  Expression Patterns
• Background Review of DNA Arrays