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Single Nucleotide Polymorphism

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Title: Single Nucleotide Polymorphism


1
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2
Single Nucleotide Polymorphism
  • ((SNP, snip

3
Human Genome
  • The human genome has 2.91 billion base pairs
  • and approximately 35.000 genes.

4
Single Nucleotide Polymorphism
  • Its the difference of one base at specific base
    pair position.
  • .e.g. from Cytosine ?Thymine
  • Adenine ? Guanine
  • One difference/1250 base pair/two unrelated
    individual
  • Of total 2.3 million differences.
  • 10-30million SNPs in the Human genome

5
Effect of SNP
  • Silent
  • Alter the function of the protein
  • Directly alter an amino acid sequence
  • indirectly alter the function of the
    regulatory sequence

6
Environmental Genome Project (EGP)
7
Environmental Genome Project (EGP)
  • It is a resequencing of the DNA
  • for detecting the polymorphic sites that are
    representing at 1 or higher in the sampled
    population.
  • the frequency of polymorphisms can vary in
    different population subgroups.

8
SNPs of Clinical significance in
  • Adverse Drug reaction
  • Disease predisposition

9
SNPs and Adverse Drug reaction
  • SNPs Altering the function of drug metabolizing
    enzymes.
  • Pharmacogenetic promise to develop individual
    medicine according to patient genotype

10
Role of SNPs in Disease predisposition
  • The Common disease are multifactorial
  • The Genetic differences between human populations
    make one population more susceptible to
    particular disease.

11
SNPs and Disease predisposition
  • Epidemiological and biomedical researches are
    looking for comparing SNPs in patients and
    healthy controls in different population
    subgroup.
  • SNPs have proven capable of locating genes
    involved in cancers, sickle cell anemia,
    psoriasis, migraine, Alzheimer and Diabetes.

12
SNPs and Cancer
13
SNPs and Cancer
  • SNPs in genes involved in DNA repair and drug
    metabolizing enzymes which responsible for
    metabolism detoxification of Carcinogens can
    act as cancer susceptibility genes
  • Through
  • increase activation of chemical
    carcinogens
  • decrease ability of cells to detoxify
    repair mutagenic damage

14
  • So
  • identification of these genotypic variations
    and their association with cancer
  • May help to
  • - Elucidate cancer etiology.
  • -predict disease risk and response to
    chemotherapy.

15
SNP and Cancer
  • There are many studies conducted over the past
    few years have identified variants alleles for
    number of drug metabolizing and DNA repair genes.
  • e.g. MTHFR (methyl tetrahydofolate
    reductasesome)
  • GST (Glutathione S-Transferases).
  • NAD(p)H quinone Oxidoreductases.
  • CytochromeP450 monooxigenases.
  • N-Acetyle Transferases.
  • change the enzyme activity with a relation to
    Cancer.

16
MTHFR gene
  • chromosome 1 (1p36.3).
  • it direct folate metabolites towards
    remethylation of homocystien and away from
    DNARNA biosynthesis.

17
  • MTHFR gene

18
MTHFR gene
  • SNPs C677T, A1298C
  • Protective Effect against DNA mutagenic damage.
  • Reduced risk of childhood ALL.

19
GST genes(GluthathhioneS-transferases)
  • Superfamily of 4 major subfamilies.
  • It catalyses the detoxification of exogenous and
    endogenous reactive substances.
  • The deletion polymorphism (GST M1 null GST T1
    null) result in absence of enzyme activity.
  • Which have increased risk of acute leukemia,
    colorectal adenocarcinoma, thyroid cancer,
    stomach, lung and pancreatic cancer in heavy
    smokers

20
NAD(p)H quinone oxidoreductase
  • locate in chromosome 16.
  • protect the cells from oxidative damage.
  • The SNP C609T results in decrease enzyme
    activity.
  • ? risk of lung cancer and acute leukemia.

21
Cytochrome P450 genes
  • It is a superfamily of genes.
  • Cytochrome monooxygenases associated in the
    metabolism of drugs, environmental pollutants and
    endogenous substances.
  • Cyp1A1 polymorphism ? risk of acute leukemia and
    postmenopausal breast cancer in early smoking
    woman.

22
N-Acetyltransferase genes
  • NAT1 NAT2 gene on chromosome 8.
  • N-acetylation (deactivation) and O-acetylation
    (activation) of aromatic and heterocyclic amine
    carcinogens.

23
N-Acetyltransferase genes
  • The slow acetylator polymorphism in NAT2
    (T341C) (C481T) SNPs associated with reduced
    enzyme activity.
  • with increased risk of
  • ALL
  • Urinary bladder cancer in smokers

24
Methods of identification of SNPs
25
Methods of identification SNPs
  • A) Detection of known SNPs
  • B) Identification of new SNPs

26
A) Detection of known SNPs
  • a) Gel-Based genotyping methods
  • 1-PCR with restriction enzyme coupled analysis.
  • 2-Amplification refractory mutation system
    (ARMS).
  • 3-Oligonucleotide ligation assay.
  • 4-Minisequencing.

27
1-PCR with restriction enzyme coupled analysis.
  • Most common .
  • Especially for known SNP that create or abolish a
    restriction site.
  • different restriction digestion pattern can be
    recognized.

28
1-PCR with restriction enzyme coupled analysis
29
2-Amplification refractory mutation system (ARMS).
  • primers matches the normal DNA sequence so
    amplification will occurs in normal individuals.

30
3-Oligonucleotide ligation assay.
  • hybridization with specific oligonucleotide
    probes

31
A) Detection of known SNPs
  • b) Non-Gel-based High throughput Genotyping
    Technologies
  • 1-hybridization using fluorescence resonance
    energy transfer detection (TaqMan genotyping.
    Molecular beacons).
  • 2-High-density chip array.

32
B)Identification of new SNPs
  • 2 steps
  • 1- Conformation-based mutation scanning.
  • 2-Direct DNA sequencing.

33
Conformation-based mutation scanning
  • Single-strand conformation polymorphism (SSCP).
  • most widely used methods.
  • Principle single strand DNA tend to fold into
    complex structure which determines the mobility
    of the DNA strand in non denaturating gel.

34
Single-strand conformation polymorphism (SSCP).
35
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