Transcript Final - Western Washington University

How are we different?
…at the DNA level.
Methods of Detection
• Mendelian Genetics,
• Direct DNA Sequencing,
• RFLP analysis,
– Restriction Fragment Length Polymorphism,
• Allele Specific Oligonucleotides,
• DNA Microarrays
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 3,000).
• five classes of polymorphisms.
Polymorphisms
• Single Base Pair Differences,
– Single Nucleotide Polymorphisms (SNPs),
• Microsatellites (short sequence repeats),
• Minisatellites (long sequence repeats),
• Deletions,
• Duplications.
RFLP Analysis
Restriction Fragment Length Polymorphism
• Variations in the length of DNA fragments
generated by restriction enzymes.
– the variations are caused by mutations that
abolish or create cutting sites,
– RFLPs are inherited in a co-dominant fashion
and can be used as genetic markers.
Single Nucleotide Polymorphisms
SNPs
Eco RI
site
…SNPs, 98% of all polymorphisms.
Allele-Specific Oligonucleotide
ASO
…short synthetic DNA probes able to
differentiate between sequences differing by
as little as 1 base pair,
--AGTAGCTGTAGCT-mismatch
no binding
--AGTAGCTGTAGCT---TCATCGACATCGA--
--TCATCGAaATCGA--
Probe with fluorescent or radioactive dCTPs.
ASO and PCR
…subject gene is amplified using PCR,
…bound to a solid substrate,
…probed with labeled ASOs.
ASO
labeled
wt, D508
Bind subject DNA
(PCR product) to
filter,
... probe with labeled
ASO.
DNA Arrays
…DNA systematically arrayed at high density,
– comparative genomics,
• DNA hybridization to DNA,
– inter- and intra-species comparisons, etc.
– virtual genomes for expression studies,
• “mRNA” hybridization to DNA (cDNA) for
expression studies,
– potential yet to be developed.
DNA Array
DNA
solid substrate
Probes/Targets
...Probes: are the tethered nucleic acids with known
sequence (ASO or cDNA),
– the DNA on the array,
...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.
1. DNA Microarrays,
–
oligonucleotide arrays,
2. DNA chips.
nucleic acid is
synthesized directly
onto on the substrate.
cDNA Microarrays
...denatured, double stranded probe 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...
DNA Chips
…oligonucleotides
systematically synthesized
in situ at high density
(probes).
Affymetrix DNA Chip
Ordered Array of ASOs
linker
molecule
...over a million different 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.
Hybridization Detection
…labeled target is hybridized to the array, unbound target is
washed away….
…fluorescent images are read by an optical scanner,
and intensities are compared using algorithms to
differentiate artifacts.
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 (+ splice
sites),
• 225 20-mers span 4.5 kb,
• 20 overlaps per 20-mer requires 4500 ASOs (grids),
plus controls (2-3 per grid).
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 photolithography, create a chip with
ASOs to identify any difference from wildtype DNA,
…match results with mutations at known
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,
– gene must be mapped, cloned and sequenced,
– DNA chips designed, and data storage and
analysis systems established.
>906,600 SNPs arrayed
Genotyping
Genome Profiling
…with 906,000 SNPs and 946,000 additional genetic
markers to measure copy gene number, genetic
profiles can be made,
…choose SNPs in or near genes involved in traits or
diseases,
– remember, linked genes are likely to travel together,
– or better, if the SNP is in an allele of interest, it can be
readily identified,
…compare profiles over large populations.
How are we different?
…at the RNA level.
Northern Analysis
DNA hybridizing to RNA,
DNA Arrays and Expression
…grid gene-specific ASOs onto the
DNA chip, or “known” cDNAs onto
microarrays,
– extract mRNA from a specific tissue,
– make fresh cDNA from the new mRNA,
– bind to the array for display.
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
Genes and Targets
• With many genome projects finished done, most,
if not all of the genes identified can be gridded,
– presently, several completely sequenced genomes have
been gridded,
•
•
•
•
•
•
H. sapiens (>20,000 genes)
Arabidopsis (>26,000 genes),
C. elegans (>22,500 genes),
Drosophila (>13,500 genes),
yeast (>6000 genes),
more,
• drug identification, fundamental research, etc.
www.affymetrix.com
Gene Expression Technologies
• General Scheme: Extract mRNA, synthesize labeled cDNA,
Hybridize with DNA on the array,
– DNA Chips (Affymetrix) and MicroArrays can measure mRNA
concentration of thousands of genes simultaneously,
– look for genes that are expressed similarly (clustering).
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,
– genome expression analysis provides a means of
monitoring drug/treatment regimes.
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,
• Comparisons of gene regulation between closely
related species,
• etc.
Monday
•Human and chimp DNA is ~98.7
similar,
•But, we differ in many and
profound ways,
•Can this difference be attributed,
at least in part, to differences in
gene expression, rather than
differences in the actual gene and
gene products?