sam snp final

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Multiplex Single NucleotidePolymorphismgenotyping byMALDI-TOF Mass Spectrometry

Prepared by :

Samir V. KalavadiaDept. of Pharmaceutics

NIPER-A.


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Introduction

A Single Nucleotide Polymorphism(SNP) is defined as a single basechange in a DNA sequence that occurs

in a significant proportion (more than1%) of a large population

SNPs are distributed across the humangenome at an approx. average of 1 SNPper 1000 bp


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In human beings, 99.9 percent bases are

same. Remaining 0.1 percent is different in every

individual.

This decides :

How a person looks,

Diseases he or she develops,

Drug Response,

Different attributes / characteristics /traits

This slight variation is what makes an


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It has been estimated that human

genome contains 10 millionpositions (locus) that have commonvariations among individuals in apopulation.

Source: Nature Genetics supplement, Vol 37, June 2005


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a or trateg es to nterrogateSNPs. Hybridization-based methods

Dynamic allele-specific hybridization(DASH)

Molecular beacons

SNP microarrays

Enzyme-based methods Restriction fragment length

polymorphism(RFLPs)

PCR-based methods

Flap endonuclease

Primer extension

5- nuclease

Post-amplification methods


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However there are about 30 (or more)

different SNP-genotyping Methods. Hence there are plenty of choices for

interrogating SNPs.

The underlying biochemistry remainsthe same.

The question is cost and throughput,

and accuracy !!!!


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Primer Extension

It involves the hybridization of a probeto the bases immediately upstream ofthe SNP nucleotide.

2 types of nucleotides are made useof : dNTPs & ddNTPs.


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With dNTPs, allele-specific probes, if they have 3bases complementary to target DNA

DNA polymerase will extend from the 3 end of the

probe

If the target DNA does not contain an allelecomplementary to the probe's 3 base

DNA polymerase will not be able to extend from the

3' end of the probe

The length of these chains can be estimated by MassSpectrometry


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Aim of the study

To develop a robust high-throughput

single-nucleotide polymorphism (SNP)

genotyping method, which applies allele-

specific extension using matrix-assisted laser

desorption/ ionization time-of-flight mass

spectrometry for determination of the base ina single-nucleotide polymorphic location.


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Experimental methods

Materials :

Human genomic DNA

HotStarTaq DNA polymerase(Qiagen) dNTPs, ddNTPs (Amersham)

ExoI & CIP (New England Biolabs)

2,4,6-THAP ;2,3,4-THAP;Genopure Kit(BrukerDaltonics)

ZiptipC18 (Millipore)


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Multiplex Primer Design

Primers for multiplex PCR were designed by

online software Primer 3 and using software

named Primer-Premier 5.0 to analyze

interactions among primers.

If the primers have strong actions with oneanother (the minimum value of G is about 10

kcal/mol), then the set is discarded.


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Multiplex PCR

3 l of genomic DNA (15 ng) was used astemplate for multiplex PCR. 6pmoles of the

forward and 6 pmol of the reverse primer

each were used in1HotStarTaq buffer, 1Q

solution with 300 lM dNTPs and 1.2U

HotStarTaq DNA polymerase in a 25-l vol.

The reaction was denatured for15 min at

95c(activated the Taq polymerase) and then

thermocycled for 30s at 94c, 40 s at 52c,

and 90 s at 68c 35 times


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CIP and ExoI digest:

1l of CIP (1.5 U/l) and 0.5l of ExoI (20

U/l) were added to the PCR reaction andincubated at 37c for 45 min.

Single-nucleotide extension reaction: 25 pmoles of the extension primers with100

MddNTPs and 1.5U thermosequenase

were added to 15l digested product above.An initial denaturing step of 3 min at 95c

was used followed by 35 circles of 30 s at

95c, 1 min at 50c, and 30s at 60c.


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Purification of extension primer

Extension reaction products were purified

using Genopure-Kit or ZiptipC18.

Mass spectrometric analysis 0.5-l aliquot of the purified sample was

mixed with 0.5l matrix(a mixture with the

molar ratios of 2:1:1 for 2,4,6-THAP (0.2M in

50% acetonitrile), 2,3,4- THAP (0.2M in 50%

acetonitrile), and diammonium hydrogen

citrate (0.3M in water)


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Samples were analyzed by using a Bruker

Reflex III MALDI-TOF mass spectrometer

(BrukerDaltonics).

Laser repetition rate : 20 Hz

Mode : Negative ion and linear.

Acceleration voltage : 19 kV

Source : Nitrogen laser (337 nm)


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Results

Thousands of parallel PCRs spanning thegenomic mutations or SNPs usually should be

performed to get a complete SNP genotyping.

Hence the technology for combining manyindividual PCRs per tube (multiplexing) was

suggested.

The scale of multiplex amplification has been

limited due to the serious primerprimer

interaction.

The wa of decreasin the rimer rimer


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Cycle conditions and components in the

multiplex PCR system were adjusted. Extension time (longer extension time),

annealing temperature (46c lower than

annealing temp used in single PCR) &amount of Taq DNA polymerase (more than

the amount in the single PCR system).

CIP and ExoI were added directly to the PCRsystem in order to digest the remaining dNTPs

and primers.


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Extension Primer Design

To give optimum MALDI-TOF performance in

terms of resolution & sensitivity, genotyping

primer lengths ideally should be kept to under

approx 40 bases.

Placing primers 2 bases apart imposes a limit

on the possible degree of multiplexing, which

can be overcome by a strategy, termed mass

tuning.

The assay design must avoid overlaps between

any unextended primer and possible extension


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The products of single-base extension purified with Genopure-Kit


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The products of single base extension purified with ZiptipC18.


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Selection of matrix for MALDI-TOF-MS.

The matrixes commonly include 3-HPA and

THA.

When 3-HPA is used as the matrix, the

crystal is in a circular pattern and leaves the

center of the well void, which makes auto

sampling difficult.

Here 2,4,6-THAP and 2,3,4-THAP as a

mixed matrix are used. It decreases the

intensity of peaks caused by depurination

& shows no big interference on the


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The throughput of 10/min is reported for

newer generation instruments such as theAutoFlex (Bruker-Daltonics, Germany)

Our multiplex assay would correspond to a

throughput of up to 60,000 loci in a 10hanalysis period.

No labeling reagent is necessary in this

method, so the cost is low.


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Conclusion

In this research a robust high-throughput

SNP genotyping method was developed.

The result indicates that this method is

potentially applicable to large-scale SNP

genotyping


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SNP A buzz word , Why ???

The discovery of SNPs promises to

revolutionize the process of disease detection.

Physicians can screen patients for susceptibilityto a disease just by analyzing their SNP

patterns. Also doctors can determine which drug is most

appropriate for patients just by checking theirSNP profiles.

The long cherished dream of Personalizedmedicinecan come true.

A day would come when patients would carry

with them their SNP profiles rather than

R f


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References[1] A.J. Brookes, The essence of SNPs, Gene 234 (1999) 177186.

[2] U. Landegren, M. Nilsson, P.Y. Kwok, Reading bits of genetic

information: Methods for single-nucleotide polymorphism analysis,

Genome Res. 8 (1998) 769776.

[3] F.S. Collins, L.D. Brooks, A. Chakravarti, A DNA polymorphismdiscovery resource for research on human genetic variation,

Genome Res. 8 (1998) 12291231.[4] D.G. Wang, J.B. Fan, C.F. Siao, A. Berno, P. Young, R. Sapolsky,

G. Ghandour, N. Perkins, E. Winchester, J. Spencer, L. Kruglyak, L.Stein, L. Hsie, T. Topaloglou, E. Hubbell, E. Robinson, M. Mittmann,M.S. Morris, N. Shen, D. Kilburn, J. Rioux, C. Nusbaum, S. Rozen,

T.J. Hudson, R. Lipshutz, M. Chee, E.S. Lander, Large-scaleidentification mapping and genotyping of single-nucleotidepolymorphism in the human genome, Science 280 (1998) 10771082.

[5] P.Y. Kwok, High-throughput genotyping assay approaches,

Pharmacogenomics 1 (2000) 95100.6 I.G. Gut Automation in enot in of sin le nucleotide


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[14] L.A. Haff, I.P. Smirnov, Multiplex genotyping of PCR productswith mass tag-labeled primers, Nucleic Acids Res. 25 (1997)37493750.

[15] P. Ross, L. Hall, I. Smirnov, L. Haff, High level multiplexgenotyping by MALDI-TOF mass spectrometry, Nat. Biotechnol.16 (1998) 13471351.

[16] A.V. Tillib, A.D. Mirzabekov, Advances in the analysis of DNAsequence variations using oligonucleotide microchip technology,Curr. Opin. Biotechnol. 12 (2001) 5358.

[17] O. Henegariu, N.A. Heerema, S.R. Dlouhy, G.H. Vance, P.H.Vogt, Multiplex PCR: Critical parameters and step-by-stepprotocol, BioTechniques 23 (1997) 504511.

[18] P. Juhasz, M.T. Roskey, I.P. Smimov, L.A. Haff, M.L.

Vestal,S.A. Martin, Applications of delayed extraction matrix-assisted laser desorption ionization time-of-flight massspectrometry to oligonucleotide analysis, Anal. Chem. 68 (1996)941946.

[19] M. Shahgholi, B.A. Garcia, N.H.L. Chiu, P.J. Heaney, K. Tang,

Sugar additives for MALDI matrices improve signal allowing thesmallest nucleotide change (A:T) in a DNA sequence to be


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dbSNPis a SNP database from National Center

for Biotechnology Information (NCBI). SNPediais a wiki-style database from a hybrid

organization.

The OMIMdatabase describes the association

between polymorphisms and, e.g., diseases intext form,

HGMDthe Human Gene Mutation Databaseprovides gene mutations causing or associated

with human inherited diseases and functionalSNPs,

while HGVbaseG2P allows users to visuallyinterrogate the actual summary-level associationdata

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