Struktural genomics. Practice

Transcription

1 Struktural genomics Practice

2 SNPs What is an SNP?

3 What is an SNP? Single nucleotide polymorphism SNPs

4 What is an SNP? Single nucleotide polymorphism What is the frequency of SNPs? SNPs

5 What is an SNP? Single nucleotide polymorphism What is the frequency of SNPs? At least 1% SNPs

6 What is an SNP? Single nucleotide polymorphism What is the frequency of SNPs? At least 1% How can we idetify SNPs? SNPs

7 What is an SNP? Single nucleotide polymorphism What is the frequency of SNPs? How can we idetify SNPs? At least 1% ASA (AS-PCR) SNPs

8 What is an SNP? Single nucleotide polymorphism What is the frequency of SNPs? How can we idetify SNPs? At least 1% ASA (AS-PCR) DNA sequencing SNPs

9 (wt) CGT AGT CCT TGA AGC TAT TAT GTA CGT AGC TGA (m) CGT AGT CCT TGA AGC TAC TAT GTA CGT AGC TGA Allele specific amplification - ASA

10 (wt) CGT AGT CCT TGA AGC TAT TAT GTA CGT AGC TGA (m) CGT AGT CCT TGA AGC TAC TAT GTA CGT AGC TGA Allele specific amplification - ASA

11 (wt) CGT AGT CCT TGA AGC TAT TAT GTA CGT AGC TGA (m) CGT AGT CCT TGA AGC TAC TAT GTA CGT AGC TGA Design a primer pair! Allele specific amplification - ASA

12 (wt) 5 CGT AGT CCT TGA AGC TAT TAT GTA CGT AGC TGA3 3 GCA TCA GGA ACT TCG ATA... ATA CAT GCA TCG ACT5 (m) 5 CGT AGT CCT TGA AGC TAC TAT GTA CGT AGC TGA3 3 GCA TCA GGA ACT TCG ATA... ATA CAT GCA TCG ACT5 How many primers do we need? Allele specific amplification - ASA

13 (wt) 5 CGT AGT CCT TGA AGC TAT TAT GTA CGT AGC TGA3 3 GCA TCA GGA ACT TCG ATA... ATA CAT GCA TCG ACT5 (m) 5 CGT AGT CCT TGA AGC TAC TAT GTA CGT AGC TGA3 3 GCA TCA GGA ACT TCG ATA... ATA CAT GCA TCG ACT5 How many primers do we need? 3 Allele specific amplification - ASA

14 (wt) 5 CGT AGT CCT TGA AGC TAT TAT GTA CGT AGC TGA3 3 GCA TCA GGA ACT TCG ATA... ATA CAT GCA TCG ACT5 (m) 5 CGT AGT CCT TGA AGC TAC TAT GTA CGT AGC TGA3 3 GCA TCA GGA ACT TCG ATA. ATA CAT GCA TCG ACT5 Allele specific amplification - ASA

15 (wt) 5 CGT AGT CCT TGA AGC TAT TAT GTA CGT AGC TGA3 3 GCA TCA GGA ACT TCG ATA... ATA CAT GCA TCG ACT5 (m) 5 CGT AGT CCT TGA AGC TAC TAT GTA CGT AGC TGA3 3 GCA TCA GGA ACT TCG ATA. ATA CAT GCA TCG ACT5 Allele specific amplification - ASA

16 (wt) CGT AAT GAA TTC AGT TGC Digest with RE

17 (m) CGT AAT GAA CTC AGT TGC There is an SNP in the RE site

18 (m) CGT AAT GAA CTC AGT TGC EcoRI akcióban Digest with RE

19 Electrophoresis

20 m wt Electrophoresis

21 Mutation in MLH1 gene (3. exon 5 end, ESE) Exon 1 Exon 2 Exon 3 ESE

22 Mutation in MLH1 gene (3. exon 5 end, ESE) Exon 1 Exon 2 Exon 3 What happens during mrna processing? What would happen if there were no mutations? ESE

23 Mutation in MLH1 gene (3. exon 5 end, ESE) Exon 1 Exon 2 Exon 3 Exon 1 Exon 2 Exon 3 ESE

24 DNA library 5 members (what is a DNA library?) What is the order of the fragments? AGT TGA GCT CGT GAA GTA CGT AGT.CGT TGC CGT GTG AAT..CGT AGT TAG TAG CTT..CTG TAC GTA CGT ATG.TGA AGT Chromosome walking by PCR

25 2.: 1.: Ligation Digestion to with a plasmid restriction endonucleases Constructing genome libraries

26 3.: 2.: Transformation Ligation to a plasmid to E. coli Constructing genome libraries

27 What is the sequence of the nucleotides? Which of the DNA bands correspond to the next DNA sequences? GGCCA GGCCATCGTTGA n A C G T GGC GGCC GGCCATC GGCCATCG GGCCATCGTTG GGCCAT GGCCATCGT GGCCATCGTT Sanger-method

28 What is the sequence of the nucleotides? Which of the DNA bands correspond to the next DNA sequences? GGCCA GGCCATCGTTGA n A C G T GGC GGCC GGCCATC GGCCATCG GGCCATCGTTG A3 G T T G C T A C C5 GGCCAT GGCCATCGT GGCCATCGTT Sanger-method

29 What is the order of the nucleotides of the template DNA? n A C G T A3 G T T G C T A C C5 Sanger-method

30 What is the order of the nucleotides of the template DNA? n A C G T A3 G T T G C T A C C5 GGTAGCAACT Sanger-method

31 What order was used by adding nucleotides? What is the order of the DNA template? Pyrosequencing

32 What order was used by adding nucleotides? G C T A What is the order of the DNA template? Pyrosequencing

33 What order was used by adding of nucleotides? G C T A What is the order of the DNA template? GCAGGCCT Pyrosequencing

34 KRAS onkogene 12p12 Member of RAS family, GTPase Encoding a 21kD protein (p21ras, or K-Ras) Function of K-Ras: molekular on/off switch It is an early player in many signal transduction pathways Analysis of KRAS gene by pyrosequencing

35 KRAS gene BIOMARKER The protein product of the unmutated KRAS gene performs an essential function in normal tissue signaling, mutated KRAS genes are potent oncogenes that play a role in many cancers A single amino acid substitution is responsible for an activating mutation. The transforming protein that results is implicated in various malignancies, including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma. Analysis of KRAS gene by pyrosequencing

36 The EGFR signaling pathway. Inhibition of EGFR can halt tumor growth, but not in the presence of activating KRAS mutations. Analysis of KRAS gene by pyrosequencing

37 Mutation in KRAS gene -> continously active gene -> cell proliferation -> cancer Common mutations: codon 12., 13., 61. Analysis of KRAS gene by pyrosequencing

38 Analysis of KRAS gene by pyrosequencing

39 Analysis of KRAS gene by pyrosequencing

40 Analysis of KRAS gene by pyrosequencing