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Gene Variations single nucleotides polymorphism & copy number variation. 刘戈飞 汕头大学医学院遗传学与细胞生物学教研室 gfliu@stu.edu.cn 075488900497 13502932022. Genetic Variations. Chromosome numbers Segmental duplications, Copy number variation Translocations Inversion Sequence Repeats

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gene variations single nucleotides polymorphism copy number variation

Gene Variationssingle nucleotides polymorphism&copy number variation

刘戈飞

汕头大学医学院遗传学与细胞生物学教研室

gfliu@stu.edu.cn

075488900497

13502932022

slide2

Genetic Variations

Chromosome numbers

Segmental duplications, Copy number variation

Translocations

Inversion

Sequence Repeats

Transposable Elements

Short deletions and insertions

Tandem Repeats

Nucleotide Insertions and Deletions (Indels)

Single Nucleotide Polymorphisms (SNPs)

Mutations

Sizable

Structural

Sequence

Minor

genetic markers
Genetic Markers
  • Morphological markers
  • Cytological markers
  • Biochemical and physiological markers
  • Molecular markers
  • 1980, RFLPs (restriction fragment length polymorphisms)
  • 1985, STRs (short tandem repeats, mini-satellites)
  • 1990s, SNPs (single nucleotide polymorphisms)
  • 2000s, CNV (copy number variation)
slide5

SNP

A C G T G T C G G T C T T A A AMaternal chromosome

A C G T G T C C G T C T T A A APaternal chromosome

Individual 1

A C G T G T C G G T C T T A A AMaternal chromosome

A C G T G T C G G T C T T A A APaternal chromosome

Individual 2

A C G T G T C C G T C T T A A AMaternal chromosome

A C G T G T C C T A C T T A A APaternal chromosome

Individual 3

The position of the SNP is indicated by the box. Individual 1 is heterozygous, while individuals 2 and 3 are homozygous.

Single nucleotide polymorphism (SNP)

在基因组中,不同个体的DNA序列上的单个碱基的差异被称作单核苷酸多态性。

slide6

Single nucleotide polymorphism (SNP)

1/1000

Estimated between any 2 individuals (3 m)

10 m

in the whole populations

snp effects

1

2

3

4

1

2

3

1

2

4

SNP Effects
  • SNPs in genes

In coding regions (possible protein structure changes)

 Synonymous substitutions (同义)

 Missense substitutions (错义)

 Nonsense substitutions (终止)

In coding and non-coding regions

 Change of gene expression (by diverse binding various factors)

Yield

Timing

Alternative splicing

  • SNPs in regulatory regions
    • Change of gene expression
  • SNPs in non-regulatory intergenic regions
    • Can be used as genetic markers
hapmap
HapMap

国际人类基因组单体型图计划

Towards

genome

variations

slide9
人类的所有群体中大约存在一千万个SNP位点,其中稀有的SNP位点的频率至少有1%。人类的所有群体中大约存在一千万个SNP位点,其中稀有的SNP位点的频率至少有1%。
  • 相邻SNPs的等位位点倾向于以一个整体遗传给后代。位于染色体上某一区域的一组相关联的SNP等位位点被称作单体型(haplotype)。
slide10
大多数染色体区域只有少数几个常见的单体型(每个具有至少5%的频率),它们代表了一个群体中人与人之间的大部分多态性。一个染色体区域可以有很多SNP位点,但是只用少数几个标签SNPs,就能够提供该区域内大多数的遗传多态模式。大多数染色体区域只有少数几个常见的单体型(每个具有至少5%的频率),它们代表了一个群体中人与人之间的大部分多态性。一个染色体区域可以有很多SNP位点,但是只用少数几个标签SNPs,就能够提供该区域内大多数的遗传多态模式。
slide11
HapMap的构建分为三个步骤:a在多个个体的DNA样品中鉴定单核苷酸多态性SNPs;b将群体中频率大于1%的那些共同遗传的相邻SNPs组合成单体型;c在单体型中找出用于识别这些单体型的标签SNPs。通过对图中的三个标签SNPs进行基因分型,研究者可以确定每个个体拥有图示的四个单体型中的哪一个。
slide12

Human genome is composed of “blocks”

We are so young!

with limited number of ancestors

with a few (thousands) of generations

with only a few recombination events

我们非常年轻

人类进化史上曾有一大瓶颈(约6-15万年前)

通过“瓶颈”的人类祖先群体很小(仅有万余人)

现代人类仅经过少数几千个时代(约3000-5600代)

“遗传重组”数目有限

slide14

Methods and technologies in SNP studies

  • Discovery (Find SNPs)
  • Validation (A common one or rare one)
  • Genotyping (Frequency in population)
  • Consideration:
  • Call rates
  • Flexibility
  • Throughput
  • Cost
slide15

Fundamental approaches

  • large-scale sequencing based:
  • genomic-alignment(GA),
  • reduced representation shotgun(RRS)
  • PCR based: common PCR
  • hybridization based: DNA chips
how to discover snps
How to discover SNPs

Genomic DNA

mRNA

RRS (reduced representation

shot-gun) library or

sampling

BAC library

cDNA library

BAC overlap

Shotgun overlap

EST overlap

Sequence overlap SNP discovery

GTTTAAATAATACTGATCA

GTTTAAATAATACTGATCA

GTTTAAATAGTACTGATCA

GTTTAAATAGTACTGATCA

slide17

Discovering SNPs by Sequencing

Phred

Phrap

Sequence

Amplify DNA

Base-calling

Contig assembly

5’

3’

Quality determination

PolyPhred

Polymorphism detection

ATAGACGATACACG

ATAGACGATACACG

ATAGACG

ATACACG

Consed

Sequence viewing

Polymorphism tagging

Analysis

Homozygotes

Heterozygote

Polymorphism reporting

Individual genotyping

Phylogenetic analysis

snp genotyping
SNP检定— Genotyping

目标:灵敏、准确、简单、高通量、低成本

Throughput

SNaPshot(ABI)、SNuPe(GE)、TaqMan(ABI)、Pyrosequencing

Fluorescence Polarization(PE)、MassArray(Sequenom)

Invader(Third Wave)、SNPlex(ABI)、

Parallele、BeadArray(Illumina)

snp screening of certain genes

Genes, Samples, Phenotypes

Primers design and PCR

-1000~-1 regulation region

5’UTR exons 3’UTR

Directly DNA sequencing

Statistical Analysis

SNP screening of certain genes
slide20
SNP raise the resolution of genetic analysis
  • Pharmacogenomics
  • Personalize medicine
slide25
Overall, the authors found 1,447 discrete, heterogeneously distributed, copy number variable regions (CNVRs), which cover 12% of the human genome. They found that 24% of CNVRs are associated with segmental duplications.
slide26
CNVRs contain different classes of functional elements.
    • many CNVs preferentially lie outside genes.
    • genes that are involved in cell-adhesion functions, sensory perception of smell and response to chemical stimuli are enriched within CNVs.
    • Conversely, cell signalling and proliferation, as well as kinase-and phosphorylationrelated categories were underrepresented among CNVs.
    • Interestingly, ultraconserved elements are strongly excluded from these regions.
slide27
CNV has effects on SNP genotype patterns. SNP has the ability to identify linked CNV.
  • Both types of variation will need to be collected and analysed systematically if we are to understand the genetic basis of human disease.
slide28
The authors call for standard assessment of CNV in all studies of the genetic basis of phenotypic variation, and for an international effort to continue to characterize and catalogue structural genomic variation.
slide29

26,628 clones

534500 SNPs

slide31

Effects of CNV

  • phenotype: modify drug response
  • predispose to or cause disease
  • polymorphism: population genetics
  • genome wide gene regulation variation
slide35

Methods to identify CNV

  • Genome-wide
    • array-based
      • array- CGH: Clone-based(1Mb), oligonucleotide-based(30kb)
      • SNP array (signal intensity, genotyping)
    • sequence-assembly comparison
  • Targeted
    • PCR-based
      • MAPH, MLPA, QMPSF: mutiplex, up to 40 regions per time
      • real-time qPCR
    • Hybridization-based
      • FISH, Southern blotting
  • Computation approaches
slide38

Methods to identify CNV: array-CGH

representational oligonucleotide microarray analysis, ROMA

slide39

Methods to identify CNV: targeted PCR-based

multiplex amplifiable probe hybridization, MAPH

slide40

Methods to identify CNV: targeted PCR-based

Multiplex ligation-dependent probe amplification, MLPA

slide41

Methods to identify CNV: targeted PCR-based

Quantitative multiplex PCR of short fluorescent fragments, QMPSF

slide44

submicrosopic

microscopic

Validation of CNV

  • Mass spectrometery: MALDI-TOF
  • real-time quantitative PCR
  • Southern blotting
  • FISH
slide45

MassArray (1)

DNA 提取

目标序列的扩增

第一次纯化和SNP位点延伸反应

Primer

点样

MALDI-TOF 质谱测定

自动序列分析及等位基因信息的获得

slide46

Allele 1

Allele 2

Unlabeled Primer (23mer)

Unlabeled Primer(23mer)

T C T

A C T

T

G

Extended Primer (24mer)

Extended Primer (26-mer)

A

A

T C T

A C T

Allele 2

Allele 1

Allele 2

Allele 1

MassArray (2)

+Enzyme

+ddATP

+dCTP/dGTP/dTTP

slide48

Identify CNV of certain genes or regions

Southern (small samples)

FISH (optional)

real-time qPCR

QMPSF

MAPH

MLPA

(large samples)