Hafta IX Genombilimde Mikrodizin Uygulamalar ve Biyoinformatik Analiz

DNA Mikroarrayler: SNP analizleri

Do. Dr. Hill zda

DNA Mikrodizin
Genomik Dizi

SNP T/G

SNP
prob = 25 baz

Perfect Match Mismatch Perfect Match Mismatch Allel B Allel A

DNA Mikrodizin
SNP C/A TAGCCATCGGTA N GTA C TCAATGATCAGCT ATCGGTAGCCAT G CAT G AGTTACTA ATCGGTAGCCAT C CAT G AGTTACTA ATCGGTAGCCAT T CAT G AGTTACTA ATCGGTAGCCAT A CAT G AGTTACTA
PM Allel MM Allel PM Allel MM Allel A A B B

DNA Mikrodizin
7 Prob ift Seti (Kuartet) SNP bana 56 olaslk Oryantasyon bana 28 Datlm yerleim
Dz zincir
5 PM A MMA PM B MMB 3 5 3

-4 -2 PM A MMA PM B MMB

-1

Ters zincir

DNA Mikrodizin
1
PMA MMA PMB MMB

2
PMA MMA PMB MMB

3
PMA MMA PMB MMB

4
PMA MMA PMB MMB

5
PMA MMA PMB MMB

6
PMA MMA PMB MMB

7
PMA MMA PMB MMB

14 kuartet deerlendirilip en iyi 10 tanesi herbir TNPyi temsil etmek zere seilir. Seilen bu 10 kuartet herbir TNPnin varln hem dz hem de ters zincirde inceler. Tek bir TNPyi sorgulamak zere toplam 40 prob kullanlmaktadr.

Tek Primer Amplifikasyonu ile Karmakln Ortadan Kaldrlmas

DNA Mikrodizin
250 ng Genomik DNA
Xba Xba Xba

RE muamelesi

Adaptrn balanmas Tek primer ile amplifikasyon 4 PCR Reaksiyonu Fragmentasyon ve iaretleme Hibridizasyon Ve Tarama

DNA Mikrodizin
Mikrosatellit # Marker znrllk # PCR Reak/rnek # PCR Primer/rnek Sre (400 rnek) Belirte bana DNA Deney bana DNA 400 10 cM (10 MB) 200 (2x multipleks) 800 > 8 hafta 40 ng 16,000 ng 10K 10,000 1 cM (1 MB) * 4 1 4-6 hafta .025 ng 250 ng

DNA Mikrodizin
Farkllklar Dizin says Enzim says Balang DNA miktar PCR Amplifikasyon Boyutu Fragmente edilecek miktar Tarama Fragmentasyon reaktifinin konsantrasyonu Algoritma ve yazlm Ana uygulama alan TNPler 100k 2 2: Xba1 ve HindIII 250 ng X2 enzim = 500 ng 250-2000bp 40ug Yksek znlrlkl tarama 10K 1 1: Xba 250 ng 250-1000bp 20ug Rutin tarama

0.06U/uL DM Algoritma ve GTT Asosiyasyon almalar Perlegen ve Kamu

0.048U/uL MPAM ve GDAS 2.0 Balant Kamu

DNA Mikrodizin

DNA Mikrodizin

DNA Mikrodizin

DNA Mikrodizin

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Resequencing Tiling Strategy

A ATCGGTAGCCATACATGAGTTACTA T ATCGGTAGCCATTCATGAGTTACTA C ATCGGTAGCCATCCATGAGTTACTA 12 bases G ATCGGTAGCCATGCATGAGTTACTA
ATCGGTAGCCATGCATGAGTTACTA CAGCT TAGCCATCGGTACGTACTCAATGAT GTCGA TAGCCATCGGTAGGTACTCAATGAT

A TAGCCATCGGTACGTACTCAATGAT T TAGCCATCGGTATGTACTCAATGAT C TAGCCATCGGTAAGTACTCAATGAT G

Resequencing Tiling Strategy

TCGGTAGCCATGAATGAGTTACTAC TCGGTAGCCATGCATGAGTTACTAC TCGGTAGCCATGGATGAGTTACTAC TCGGTAGCCATGTATGAGTTACTAC ATCGGTAGCCATGCATGAGTTACTA CAGCT TAGCCATCGGTACGTACTCAATGAT GTCGA AGCCATCGGTAGATACTCAATGATG AGCCATCGGTAGCTACTCAATGATG AGCCATCGGTAGGTACTCAATGATG AGCCATCGGTAGTTACTCAATGATG

Resequencing Tiling Strategy

CGGTAGCCATGCATGAGTTACTACA CGGTAGCCATGCCTGAGTTACTACA CGGTAGCCATGCGTGAGTTACTACA CGGTAGCCATGCTTGAGTTACTACA ATCGGTAGCCATGCATGAGTTACTA CAGCT TAGCCATCGGTACGTACTCAATGAT GTCGA GCCATCGGTACGAACTCAATGATGT GCCATCGGTACGCACTCAATGATGT GCCATCGGTACGGACTCAATGATGT GCCATCGGTACGTACTCAATGATGT

Resequencing Tiling Strategy

GGTAGCCATGCAAGAGTTACTACAG GGTAGCCATGCACGAGTTACTACAG GGTAGCCATGCAGGAGTTACTACAG GGTAGCCATGCATGAGTTACTACAG ATCGGTAGCCATGCATGAGTTACTA CAGCT TAGCCATCGGTACGTACTCAATGAT GTCGA CCATCGGTACGTACTCAATGATGTC CCATCGGTACGTCCTCAATGATGTC CCATCGGTACGTGCTCAATGATGTC CCATCGGTACGTTCTCAATGATGTC

Resequencing Tiling Strategy

GTAGCCATGCATAAGTTACTACAGC GTAGCCATGCATCAGTTACTACAGC GTAGCCATGCATGAGTTACTACAGC GTAGCCATGCATTAGTTACTACAGC ATCGGTAGCCATGCATGAGTTACTA CAGCT TAGCCATCGGTACGTACTCAATGAT GTCGA CATCGGTACGTAATCAATGATGTCG CATCGGTACGTACTCAATGATGTCG CATCGGTACGTAGTCAATGATGTCG CATCGGTACGTATTCAATGATGTCG

Resequencing Tiling Strategy

TAGCCATGCATGAGTTACTACAGCT TAGCCATGCATGCGTTACTACAGCT TAGCCATGCATGGGTTACTACAGCT TAGCCATGCATGTGTTACTACAGCT ATCGGTAGCCATGCATGAGTTACTA CAGCT TAGCCATCGGTACGTACTCAATGAT GTCGA ATCGGTACGTACACAATGATGTCGA ATCGGTACGTACCCAATGATGTCGA ATCGGTACGTACGCAATGATGTCGA ATCGGTACGTATTCAATGATGTCGA

A C
Dz zincir

G T

T C G C A

CustomSeq Resequencing Dizinleri

A C G T

AGCGT
A C G T

ACCGT
ip bana 30 kb ift zincirli DNA dizilimi okunabiliyor. Toplam 60 kb.

CustomSeq Deney Protokol

Kontrol Kalp/Primerler Genomik DNA

Ksa lekli PCR

Uzun lekli PCR

Miktar tayini-rneklerin Kartrlmas

Fragmentasyon reaktifi

Fragment aret

Hibridizasyon iin oligo kontrolleri

Hibridizasyon

Veri analizi

Deney Ak
lgilenilen genlerin belirlenmesi

Kartrma ve Fragmentasyon Fragmentasyon

DNA fragmentleri fragmentleri PCR rnleri aretleme

Genomik Genomik DNA

Geceboyu hibridizasyon

B B B B B B

Tarama

Ykama stasyonu: stasyonu: Ykama/Boyama

Sondan iaretli fragmentler

Affymetrix CustomSeq Arrays

2.5 Days
Biological Sample
Probe Array

>chr21:29595444-29636248 gtggcatgatcttggctcactgcagttcaagcaattctcctgccttagc cccctgagtagctgggattacaggtgcctgctaccaccctcagctaat tttttgtatttttaatagagacggggtttcaccatgttggccaggctggtc tcgaactcctgacctcgtgattcgtctgcctcggccttccaaagtgctg ggagtacaggcatgagccaccgcacccggcctgtatatcttttttaaa aggaaaagaacatatcccagaagtttccaactgagtttccctctgggt caataccagaattgggtgggggtcttcagctcacccctaaactcatca ttgataagcagaatgaatgaccatggctggtttagaca.

~30 Kb
Wash & Stain Scan

DNA Analiz Yazlm

Cihaz Kontrol .DAT dosyasnn oluturulmas .CEL dosyasnn oluturulmas .CHP dosyasnn oluturulmas .CHP veri karlatrlmas GeneChip DNA Analysis Software 2.0 (GDAS) GCOS