Download Single Nucleotide Polymorphism (SNP) 分析與應用

Single Nucleotide Polymorphism (單一核苷酸多型性) Single Nucleotide Polymorphism (SNP) 分析與應用
林照雄
國立陽明大學
生命科學系暨基因體科學研究所
生物醫學資訊研究所
生物藥學資訊研究所
Nature (2001) 409:923-933.
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Central dogma of life Single Nucleotide Polymorphism (單一核苷酸多型性) • Human Genome Project produces numerous sequence data, and it has
become evident that there is substantial variation in DNA sequences
between two individuals at many points throughout the genome –
genome variation.
• Most commonly, sequence variation occurs at discrete, single‐nucleotide
positions referred to as single nucleotide polymorphisms (SNPs), which
are estimated to occur at a frequency of approximately one per 1000
nucleotides.
• SNPs are biallelic polymorphisms, that is, the nucleotide identity at these
polymorphic positions is generally constrained to one of two possibilities
in humans,
humans rather than the four nucleotide possibilities that could occur,
occur
in principle. For a variation to be considered a SNP, it must occur in at
least 1% of the population.
• SNPs have great potential for use in genetic‐mapping studies, which
locate and characterize genes that are important in human disease and
g
ffunction.
biological
‐ Biomedical researches link something to disease risks & response to therapy
The Lancet (2000) 356:1749-1756.
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Trends in Biotechnology (2000) 18:77-83.
http://www.ornl.gov/sci/techresources/Human_Genome/faq/snps.shtml - SNP Fact Sheet
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Adjacent SNPs that are inherited together are compiled into ʺhaplotypesʺ Single Nucleotide Polymorphism (單一核苷酸多型性) • SNPs are useful for finding genes that contribute to disease, in two ways.
Some SNP alleles are the actual DNA sequence variants that cause
differences in gene function or regulation that directly contribute to
disease processes. Most SNP alleles, however, probably contribute little
to disease. They are useful as genetic markers that can be used to find
the functional SNPs because of associations between the marker SNPs
and the functional SNPs.
• SNPs of various types can change the function or the regulation and
expression of a protein. The most obvious type is a nonsynonymous SNP,
where the alleles differ in the amino acid of the protein product. Some
polymorphisms
y
p
at splice
p
sites,, and result in variant p
proteins that
SNPs are p
differ in the exons they contain. Some SNPs are in promoter regions and
are reported to affect the regulation and expression of proteins. When
SNPs are associated with other SNPs,
SNPs then many SNPs,
SNPs in exons,
exons introns,
introns
and other noncoding regions, may all be associated with a disease or
phenotype, even though only one or a few may directly affect the
phenotype
phenotype.
Methods in Molecular Biology (2003) page 1, vol 212 - Single Nucleotide Polymorphisms, Humana Press
http://snp.cshl.org/
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International HapMap Project 6
Single Nucleotide Polymorphism (單一核苷酸多型性) • SNPs and other less common sequence variants are the ultimate basis for
genetic differences among individuals, and thus the basis for most
genetic contributions to disease.
disease To make good use of SNPs for finding
genes related to disease and studying their function, better and cheaper
technological methods are needed for discovering SNPs, for genotyping
them in many individuals,
individuals for finding their frequencies in pooled samples,
samples
and for discerning haplotypes. New statistical methods are needed to
analyze linkage and association in large‐scale studies, to relate
h l
haplotypes
and
d the
h diploid
d l d genotypes they
h form
f
to disease
d
risk,
k and
d to
elucidate the interactions among genes and between genes and the
environment.
• For SNPs to realize their full potential in genetic analysis, thousands of
different SNP loci must be screened in a rapid, accurate and cost‐
p
y is a p
promising
g tool ffor
effective manner. MALDI–TOF mass spectrometry
the high‐throughput screening of SNPs, with future prospects for use in
genetic analysis.
http://snp.cshl.org/
Methods in Molecular Biology (2003) page 1, vol 212 - Single Nucleotide Polymorphisms, Humana Press
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Identification of SNP
Identification of SNP
Genotyping SNPs With Molecular Beacons
Single‐strand conformation polymorphism (SSCP) + separation method
Methods in Molecular Biology (2003) page 37, vol 212 - Single Nucleotide Polymorphisms, Humana Press
Methods in Molecular Biology (2003) page 111, vol 212 - Single Nucleotide Polymorphisms, Humana Press
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Identification of SNP
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MALDI‐TOF mass spectrometry Strategies for SNP genotyping by primer extension using microarrays
Matrix‐Assisted Laser Desorption/Ionization – Time Of Flight
mass analysis linear type MH+
reflectron
ionization Nature Reviews Genetics (2001) 2, 930-942.
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Mass spectrometry is a powerful analytical technique with detection sensitivity at
for a compound of mass 1000 Daltons) and accuracy at 0.01% or 5 ppm.
10‐12g
(10‐15 moles
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MALDI‐TOF mass spectrometry MALDI‐TOF mass spectrometry Matrix
Mass spectrum
3-Hydroxypicolinic
y
yp
acid (HPA)
(
)
 Cyano 4 hydroxy
‐Cyano‐4‐hydroxy‐
cinnamic acid (CHCA)
Peptides<10kDa
Sinapinic Acid (SA)
Proteins >10kDa
N t lC b h d t
2,5‐Dihydroxybenzoic acid 2
5 Dih d
b
i
id Neutral Carbohydrates,
Synthetic Polymers
(DHB)
Super DHB ( + 10% 5‐
methoxysalicylic acid )
Proteins,
Glycosylated proteins
3‐Hydroxypicolinic acid
(HPA)
Oligonucleotides
2‐(4‐hydroxy‐phenylazo)
benzoic acid (HABA)
Proteins,
Oligosaccharides
Mol. Cell. Proteomics (2003) 422:117-126.
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Advantages using MALDI‐TOF MS for nucleic acid analysis
SNP analysis using MALDI‐TOF MS
• Mass spectrometry is fast; ionization, separation by size, and detection of nucleic
acids takes milliseconds to complete. Because signals from multiple laser pulses
(~20–100 pulses) are usually averaged to obtain a final mass spectrum, the total
analysis time can take as little as ten seconds.
seconds
• By contrast, conventional electrophoretic methods for separating and detecting
nucleic acids can take hours to complete.
• The results of mass spectrometry
p
y are absolute,, beingg based on the intrinsic
property of the mass‐to‐charge ratio (m/z). This is inherently more accurate than
electrophoresis‐based or hybridizationarray‐based methods, which are both
susceptible to complications from secondary‐structure formation in nucleic acids.
• Furthermore,
h
the
h absolute
b l
nature off detection,
d
i
combined
bi d with
i h the
h detection
d
i off
predominantly singly charged molecular ions, makes the analysis of complex
mixtures by MALDI–TOF MS possible.
• The complete automation of all steps,
steps from sample preparation through to the
acquisition and processing of the data, is feasible, giving MALDI–TOF MS great
potential for high‐throughput nucleic acid analysis applications.
DNA sequencing for SNP genotyping
A: 135 Da
G: 151 Da
C: 111 Da
PCR reactions with ddNTP for sequencing previously unknown single‐nucleotide i l
k
i l
l id
substitution mutations Non‐extended primer
‐ There is a loss of signal intensity and mass resolution with increasing DNA size; this has proven to be the major current limitation to DNA sequencing by MALDI TOF MS
limitation to DNA sequencing by MALDI–TOF MS
T: 126 Da
Trends in Biotechnology (2000) 18:77-83.
Nature Biotechnology (1998) 16:381-384.
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SNP analysis using MALDI‐TOF MS
SNP analysis using MALDI‐TOF MS
Minisequencing
Minisequencing for multiplex genotyping
Mass‐tagging ddNTPs
Genome Research (1997) 7:378-388.
Nature Biotechnology (1998) 16:1347-1351.
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SNP analysis using MALDI‐TOF MS
SNP analysis using MALDI‐TOF MS
MassEXTEND assay A nucleotide depletion primer extension assay homozygous
A
heterozygous
GA
No dGTP
Journal of Proteome Research (2004) 3:218-227.
homozygous
Rapid Communication in Mass Spectrometry (2004) 18:2249-2254.
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SNP analysis using MALDI‐TOF MS
SNP analysis using MALDI‐TOF MS
Peptide nucleic‐acid (PNA) hybridization (
)
probes • The neutral backbone increased thermal stability of the resulting duplex
Detection of SNPs in double‐stranded DNA by using exonuclease III/nuclease /
S1/PNA system •
•
•
•
PNA has the ability to hybridize under low ionic strength conditions
PNA has an increased hybridization specificity for complementary DNA sequences
PNA has an increased hybridization specificity for complementary DNA sequences
PNA oligomers are useful as allele‐specific hybridization probes
PNA is easily analyzed by MALDI‐TOF MS
Nature Biotechnology (1997) 15:1368-1372.
Nucleic Acid Research (2004) 32:e42.
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SNP analysis using MALDI‐TOF MS
SNP analysis using MALDI‐TOF MS
SNP genotyping using invader assay and MALDI‐TOF MS
SNP genotyping using RNA
Indel: insertion/deletion
Indel: insertion/deletion
Human ApoE nt 5208‐5244
PNAS (1999) 96:6301-6306.
Journal of American Society of Mass Spectrometry (2006) 17:3-8.
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SNP analysis using MALDI‐TOF MS
SNP analysis using MALDI‐TOF MS
SNP genotyping using RNA
SNP genotyping using RNA
1
2
3
4
5
RNAdel
RNAdel
RNAc
RNAc
RNAA
RNAA
RNAcc
RNAccc
3+5
3+4
1+2
3+4+5
2+3+5
3+4+2
1+2+3+5
3+4+2+5
RNAcc
RNAccc
Journal of American Society of Mass Spectrometry (2006) 17:3-8.
Journal of American Society of Mass Spectrometry (2006) 17:3-8.
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Application – Hepatitis C Virus Genotyping
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Application – Hepatitis C Virus Genotyping
Determination off the
h hepatitis
h
C virus (HCV)
(
) genotype has
h become
b
accepted
d as the
h
standard procedure in laboratory practice. Genotype assignment helps in disease
prognosis and assists in establishing the appropriate duration of treatment.
HC1a
HC21
HC1b
HC21
HC2a
HC21
HC3a
5’ UTR
HC2
HC4
HC2
HC5
HC1a
HC21
HC1b
HC1a
HC1b
HC2a
HC3a
HC4
HC5
HC1
HC2a
HC1
HC3a
HC1
HC4
HC1
HC5
HC1
HC1
HC1
HC1
HC1
HC1
HC1
HC1a
HC1b
HC2a
HC3a
HC4
HC5
HC21
HC21
HC21
HC2
HC2
HC21
HC1
Journal of Clinical Microbiology (2005) 43:2810-2815.
Journal of Clinical Microbiology (2005) 43:2810-2815.
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Application – Hepatitis C Virus Genotyping
nonextended
Application – Hepatitis C Virus Genotyping
H1a
H1b
Journal of Clinical Microbiology (2005) 43:2810-2815.
H2a
Journal of Clinical Microbiology (2005) 43:2810-2815.
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Application – Hepatitis C Virus Genotyping
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Application – Hepatitis C Virus Genotyping
HCV typing results for 69 HCV‐positive sera obtained by the minisequencing reaction y
p
y,
p
p
,
q
g
followed by mass spectrometry, allele‐specific amplification, and direct sequencing
H3a
H4
Journal of Clinical Microbiology (2005) 43:2810-2815.
Journal of Clinical Microbiology (2005) 43:2810-2815.
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Application – association of SNP with diseases Application – association of haplotype with diseases Pharmacogenomics (2003) 3:17‐26.
(2003) 3:17‐26
J Mol Med (2001) 79:732‐737.
Cancer Research (2002) 62:4992‐4995.
h(
)
PNAS (2005) 97:10483‐10488.
Nature Genetics (2003) 35:349‐10488.
Journal of Clinical Microbiology (2005) 43:2810-2815.
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