Download DNA-Arrays

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.
How are we different?
…at the DNA level.
Southern Analysis
DNA hybridizing to DNA,
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.
Polymorphisms
• Single Base Pair Differences,
• Microsatellites (short sequence repeats),
• Minisatellites (long sequence repeats),
• Deletions,
• Duplications.
Single Base Differences
5’-------G-A-A-T-T-C--------3’
| | | | | |
3’-------C-T-T-A-A-G--------5’
A to G mutation
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).
Point Mutations
• Chemical Mutagens,
• UV-light,
• other natural and synthetic causes.
Detecting Point Mutations
• Direct DNA Sequencing,
• RFLP analysis,
• Alleles Specific Oligonucleotides...
Allele-Specific Oligonucleotides
…short synthetic DNA probes able to
differentiate between sequences differing by
as little as 1 base pair,
--AGTAGCTaTAGCT-mismatch
no binding
--AGTAGCTGTAGCT---TCATCGACATCGA--
--TCATCGACATCGA--
Probe with fluorescent or radioactive CTPs.
Complementary, binding.
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.
ASO and PCR
…subject gene is amplified using PCR,
…bound to a solid substrate,
…probed with ASOs.
Put DNA
on filter,
probe with
the ASO.
Microsattelites
…DNA element composed of 15-100 tandem
repeats of one-, two- or three base
sequences,
AAAAAAAAAAAAAAAAAAAAAAAAAAA
CACACACACACACACACACACACACACA
CATCATCATCATCATCATCATCATCATCAT
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, doesn’t occur too often.
DNA Polymerase Slippage
replication incorporates
repeats...
replications
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.
Unequal Crossing Over
...minisattelite mutation frequency, 10-3.
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.
Detecting Polymorphisms
• RFLPs Classical: restriction fragment length polymorphisms,
– Advantage: don’t need sequence information,
– Disadvantage: need lots of DNA and radioactive detection, labor and time
dependent,
– Major Use: genotyping in absence of sequence info.
Detecting Polymorphisms
• RFLPs PCR: restriction fragment length
polymorphisms,
– Advantage: don’t need lots of DNA or radioactivity,
– Disadvantage: labor, time dependent,
– Major Use: genotyping.
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.
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.
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.
Use of multiple loci provides a definite DNA fingerprint.
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.
RAPDs
…random amplification of polymorphic DNA;
...the use of non-specific DNA sequences to
describe a specific genome.
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.
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 >3000 loci.
Random Distribution
…this experiment would generate three PCR products.
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.
Deletions, Duplications
…rare events,
– karyotyping,
– SSCPs,
– PCR (look for product size differences),
– direct sequencing of DNA,
– etc.
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 molecule’s
conformation.
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,
– don’t need to know sequence of mutant alleles,
• Disadvantage:
– 90% maximum detection rate.
Genetic Disease Detection
• RFLPs,
• ASOs,
• other PCR protocols,
…determine genotypes that result in diseases
directly,
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.
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.
Arrays
DNA Chip:
oligonucleotides, up to
1000s kb fragments.
solid substrate
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.
DNA-Probes
– cDNA arrays, DNA arrays,
nucleic acid is spotted
onto the substrate.
• DNA Microarrays,
– oligonucleotide arrays,
• DNA chips.
nucleic acid is
synthesized directly
onto on the substrate.
DNA Chips
…oligonucleotides
systematically synthesized
in situ at high density.
Affymetrix DNA Chip
Allele-Specific Oligonucleotides
(DNA Chips)
--AGTAGCTaTAGCT---AGTAGCTGTAGCT--
--TCATCGACATCGA--
mismatch
no binding
--TCATCGACATCGA--
…allele specific oligonucleotides (ASOs)
recognize single base pair differences in
DNA sequences.
Ordered Array of ASOs
linker
molecule
...over a million ASOs and controls can be gridded per cm2.
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.
QuickTime™ and a
Animation decompressor
are needed to see this picture.
Targets
...fluorescent targets,
– genomic DNA,
– cDNA, mRNA or cRNA
for expression studies,
…targets are washed over the chip for hybridization.
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,
quill technology...
Hybridization Detection
…fluorescent images are read by an optical scanner,
and intensities are compared using algorithms to
differentiate artifacts.
DNA Chip
Probe?
Oligos
DNA Microarray
Probe?
cDNAs
Target? mRNA Transcripts Under Two Conditions.
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.
Cystic fibrosis Detection
• Create a DNA chip with ASOs for wildtype 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.
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.
Cystic fibrosis Chip
…using photlithography, create a chip with
ASOs to identify any difference from wildtype DNA,
…match results with mutations at know
deleterious loci,
…catalog new deleterious loci.
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.
+
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.
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.
How are we different?
…at the RNA level.
Northern Analysis
DNA hybridizing to DNA,
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.
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.,
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.
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.
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
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.
Friday
• Intra- and Interspecies Variation in Primate
Gene Expression Patterns
• Background: Review of DNA Arrays