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Lecture 6. Functional Genomics:
DNA microarrays and re-sequencing
individual genomes by hybridization
Goals of Functional
Genomics:
1)DNA
2)RNA
3) Protein
4) Whole organism
5) Society
Lander, E. 1996. The New Genomics: Global
Views of Biology. Science 274: 536-539.
1. DNA level:
a) Systematic identification of all common variants
in human genes, both the coding
and non-coding regions.
These are the "isotopes" to gene "elements"
b) resequencing of entire genomes
of individuals
c) comparison of fully sequenced genomes of
related (and unrelated) species
EG: man and chimp
This requires sequencing of many genomes.
Defining the COMMON DIFFERENCES
within the Human Population
Polymorphism: a variant in sequence that occurs in >1% of
the population. Can be a single nucleotide change, a deletion
or insertion.
Mutation: a variant that occurs at a frequency of <1% and that
is responsible for a disease in the human population.
Mutations in general have occurred at a more recent
evolutionary time than polymorphisms.
The most common variations among individual humans
are single nucleotide polymorphisms (SNPs), which occur
approximately once every 500 to 2000 bases. “Isotopes” to
normal base “elements”.
Why identify human polymorphisms?
Human polymorphisms, including SNPs, are associated
with a higher risk for common human genetic diseases, for
example, heart disease, diabetes, alzheimers disease,
osteoporosis and cancer.
Examples of SNPs identified by studying
one gene at a time (age of cloning approach):
-leukocyte antigen complex : type I diabetes
-ApoE4 allele : Alzheimer's disease
-insulin promoter polymorphism: type I diabetes
-Collagen1A1 gene polymorphism: osteoporosis.
These common diseases are complex, likely involving many genes, and
different combinations of gene isotopes and elements will present different
risks of disease, different prognoses, and, perhaps eventually, even different
treatments
Genome-wide surveys of polymorphisms in affected
individuals might reveal such combinations
Molecular Markers can be linked to a phenotypic trait
Re-sequencing the genome of individuals will be required
to identify combinations of gene polymorphisms
contributing to common human diseases.
Phase I: Draft Sequence of Human Genome
(5-7 Individuals; capillary sequencers)
Phase II: Identification of All Common Polymorphisms
(~30 individuals, capillary sequencers)
Phase III: Comparison of a Group of Affected Individuals
(e.g., heart disease patients) with normal siblings
(~180-200 sib pairs; Microarrays)
Phase IV: Survey of large populations (Microarrays)
Double-stranded DNA can be MELTED and REANNEALED
T(M)=temperature at which 1/2 of a DNA sequence of known composition will
be denatured (single stranded): directly proportional to G:C content
T(M) Depends on the STRINGENCY under which it is measured
T(M) of the same DNA
determined under
different conditions
Under Stringent Conditions, Hybridization
of 2 DNA strands is a measure of IDENTITY
Complete hybridization=identical sequence
A)
5’TCGGATCGACTT3’
HYBRIDIZATION=
Identical (complementary) sequence
3’AGCCTAGCTGAA5’
B)
5’TCGGATCGACTT3’
NO HYBRIDIZATION=
Sequence Difference(s)
3’AGCCTAACTGAA5’
Simple Example of Sequencing by Hybridization:
Consider the 12-mer TCGGATCGACTT.
Simple Example of Sequencing by Hybridization:
Consider the 12-mer TCGGATCGACTT.
Hybridize this 12-mer to a library of 8-mers of all possible
sequences (48 possible unique combinations, or 65,536
oligonucleotides), one at a time.
Simple Example of Sequencing by Hybridization:
Consider the 12-mer TCGGATCGACTT.
Hybridize this 12-mer to a library of 8-mers of all possible
sequences (48 possible unique combinations, or 65,536
oligonucleotides) one at a time.
If high stringency hybridization is used, only 5 overlapping
8-mers (out of 65,536) will hybridize:
Simple Example of Sequencing by Hybridization:
Consider the 12-mer TCGGATCGACTT.
Hybridize this 12-mer to a library of 8-mers of all possible
sequences (48 possible unique combinations, or 65,536
oligonucleotides) one at a time.
If high stringency hybridization is used, only 5 overlapping
8-mers (out of 65,536) will hybridize:
TCGGATCG
CGGATCGA
GGATCGAC
GATCGACT
ATCGACTT
Simple Example of Sequencing by Hybridization:
Consider the 12-mer TCGGATCGACTT.
Hybridize this 12-mer to a library of 8-mers of all possible
sequences (48 possible unique combinations, or 65,536
oligonucleotides) one at a time.
If high stringency hybridization is used, only 5 overlapping
8-mers (out of 65,536) will hybridize:
TCGGATCG
CGGATCGA
GGATCGAC
GATCGACT
ATCGACTT
TCGGATCGACTT
Alignment of these 5 gives the sequence of the 8-mer.
Simple Example of Sequencing by Hybridization:
Consider the 12-mer TCGGATCGACTT.
Hybridize this 12-mer to a library of 8-mers of all possible
sequences (48 possible unique combinations, or 65,536
oligonucleotides) one at a time.
If high stringency hybridization is used, only 5 overlapping
8-mers (out of 65,536) will hybridize:
TCGGATCG
CGGATCGA
GGATCGAC
GATCGACT
ATCGACTT
TCGGATCGACTT
Alignment of these 5 gives the sequence of the 8-mer.
If all 65,536 oligos are each synthesized on a single glass
chip, then a single hybridization reaction can be used to
determine the sequence of the 8-mer.
Synthesis of Oligonucleotides on Solid Supports
Multiple Oligonucleotides synthesized in situ on glass slides
using light directed combinatorial chemistry
In the end, 70,000 copies of
oligo synthesized per each slot
Pease, Solas, Sullivan, Cronin, Holmes, and Fodor. 1994. Light-generated
oligonucleotide arrays for rapid DNA sequence analysis. PNAS USA 91:5022-5026
Hybridization to a DNA chip is temp. dependent
Hybridization to a DNA chip is sequence specific:
Detection of a single 8-mer by hybridization to an array
of 256 octomers
Format for the DNA sequencing chip:
overlapping oligonucleotides that vary at the center position.
Wild-type
Fragmented, labeled cDNA
from HIV isolate
1500 bp of hymn HIV pol and protease genes represented
on a single array with polymorphism at every position scored
Example: Detecting a mutation in human mitochondrial DNA
Wild-type sequence
Mutated sequence
Step 3-4: Genome-wide surveys of polymorphisms
SNPs are defined, now design chips to look
at only the regions with knwn SNPs
(SNP
Chips)
Wang DG., et al. (1998). Large-scale identification, mapping,
and genotyping of single-nucleotide polymorphisms
in the human genome. Science 280:1077-1082.
Example of a SNP Chip
Use mulitplex
PCR to
amplify many
possible SNP
sites
from a single
individual;
label and
hybridize
to chips
designed
to detect the
known
SNPs