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Genomics & Biotechnology

Genomics & Biotechnology. Michael D. Kane, PhD Asst. Professor, Department of Computer & Information Technology Lead Genomic Scientist, Bindley Bioscience Center Purdue University Adjunct Asst. Professor of Pharmacology Ohio Northern University. Genomics Review

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Genomics & Biotechnology

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  1. Genomics & Biotechnology Michael D. Kane, PhD Asst. Professor, Department of Computer & Information Technology Lead Genomic Scientist, Bindley Bioscience Center Purdue University Adjunct Asst. Professor of Pharmacology Ohio Northern University

  2. Genomics Review • Single Nucleotide Polymorphisms (SNPs) • Basics of DNA Detection • SNP Discovery • SNP Detection • Biotechnologies • Data Formats • Genomic Data serving as Clinical Decision Support

  3. Genomics Review DNA is Information Storage

  4. Genomics Review “Zipped Files” Decompression “Executable Files”

  5. Genomics Review DNA is Double Stranded – One strand is the “coding strand” and the other strand is there to stabilize the DNA sequence when not in use. Double-stranded DNA is very durable in our environment.

  6. Genomics Review DNA is Double Stranded… Anti-parallel Configuration Top strand is ALWAYS written 5’ to 3’ When DNA is written in file, top strand is represented and bottom strand is assumed. 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ AGTCGTGATCTGCTAAATGTCTCGAAGTTCGATGCTAG |||||||||||||||||||||||||||||||||||||| TCAGCACTAGACGATTTACAGAGCTTCAAGATACGATC Courier font is preferred for writing sequence data since letter spacing is independent of character content.

  7. >gi|1924939|emb|X98411.1|HSMYOSIE Homo sapiens partial mRNA for myosin-IF CAGGAGAAGCTGACCAGCCGCAAGATGGACAGCCGCTGGGGCGGGCGCAGCGAGTCCATCAATGTGACCC TCAACGTGGAGCAGGCAGCCTACACCCGTGATGCCCTGGCCAAGGGGCTCTATGCCCGCCTCTTCGACTT CCTCGTGGAGGCCATCAACCGTGCTATGCAGAAACCCCAGGAAGAGTACAGCATCGGTGTGCTGGACATT TACGGCTTCGAGATCTTCCAGAAAAATGGCTTCGAGCAGTTTTGCATCAACTTCGTCAATGAGAAGCTGC AGCAAATCTTTATCGAACTTACCCTGAAGGCCGAGCAGGAGGAGTATGTGCAGGAAGGCATCCGCTGGAC TCCAATCCAGTACTTCAACAACAAGGTCGTCTGTGACCTCATCGAAAACAAGCTGAGCCCCCCAGGCATC ATGAGCGTCTTGGACGACGTGTGCGCCACCATGCACGCCACGGGCGGGGGAGCAGACCAGACACTGCTGC AGAAGCTGCAGGCGGCTGTGGGGACCCACGAGCATTTCAACAGCTGGAGCGCCGGCTTCGTCATCCACCA CTACGCTGGCAAGGTCTCCTACGACGTCAGCGGCTTCTGCGAGAGGAACCGAGACGTTCTCTTCTCCGAC CTCATAGAGCTGATGCAGTCCAGTGACCAGGCCTTCCTCCGGATGCTCTTCCCCGAGAAGCTGGATGGAG ACAAGAAGGGGCGCCCCAGCACCGCCGGCTCCAAGATCAAGAAACAAGCCAACGACCTGGTGGCCACACT GATGAGGTGCACACCCCACTACATCCGCTGCATCAAACCCAACGAGACCAAGCACGCCCGAGACTGGGAG GAGAACAGAGTCCAGCACCAGGTGGAATACCTGGGCCTGAAGGAAAACATCAGGGTGCGCAGAGCCGGCT TCGCCTACCGCCGCCAGTTCGCCAAATTCCTGCAGAGGTATGCCATTCTGACCCCCGAGACGTGGCCGCG GTGGCGTGGGGACGAACGCCAGGGCGTCCAGCACCTGCTTCGGGCGGTCAACATGGAGCCCGACCAGTAC CAGATGGGGAGCACCAAGGTCTTTGTCAAGAACCCAGAGTCGCTTTTCCTCCTGGAGGAGGTGCGAGAGC GAAAGTTCGATGGCTTTGCCCGAACCATCCAGAAGGCCTGGCGGCGCCACGTGGCTGTCCGGAAGTACGA GGAGATGCGGGAGGAAGCTTCCAACATCCTGCTGAACAAGAAGGAGCGGAGGCGCAACAGCATCAATCGG AACTTCGTCGGGGACTACCTGGGGCTGGAGGAGCGGCCCGAGCTGCGTCAGTTCCTGGGCAAGAAGGAGC GGGTGGACTTCGCCGATTCGGTCACCAAGTACGACCGCCGCTTCAAGCCCATCAAGCGGGACTTGATCCT GACGCCCAAGTGTGTGTATGTGATTGGGCGAGAGAAGATGAAGAAGGGACCTGAGAAAGGTCCAGTGTGT GAAATCTTGAAGAAGAAATTGGACATCCAGGCTCTGCGGGGGGTCTCCCTCAGCACGCGACAGGACGACT TCTTCATCCTCCAAGAGGATGCCGCCGACAGCTTCCTGGAGAGCGTCTTCAAGACCGAGTTTGTCAGCCT TCTGTGCAAGCGCTTCGAGGAGGCGACGCGGAGGCCCCTGCCCCTCACCTTCAGCGACACACTACAGTTT CGGGTGAAGAAGGAGGGCTGGGGCGGTGGCGGCACCCGCAGCGTCACCTTCTCCCGCGGCTTCGGCGACT TGGCAGTGCTCAAGGTTGGCGGTCGGACCCTCACGGTCAGCGTGGGCGATGGGCTGCCCAAGAACTCCAA GCCTACCGGAAAGGGATTGGCCAAGGGTAAACCTCGGAGGTCGTCCCAAGCCCCTACCCGGGCGGCCCCT GGCGCCCCCCAAGGCATGGATCGAAATGGGGCCCCCCTCTGCCCACAGGGGGGGGCCCCCTGCCCCCTGG AGAAATTCATTTGGCCCAGGGGGCACCCACAGGCCTCCCCGGCCCTCCGTCCACATCCCTGGGATGCCAG CAGACGACCCCGGGCACGTCCGCCCTCAGAGCACAACACAGAATTCCTCAACGTGCCTGACCAGGGGATG GCCGGCATGCAGAGGAAGCGCAGCGTGGGGCAACGGCCAGTGCCTGTGGGCCGACCCAAGCCCCAGCCTC GGACACATGGTCCCAGGTGCCGGGCCCTATACCAGTACGTGGGCCAAGATGTGGACGAGCTGAGCTTCAA CGTGAACGAGGTCATTGAGATCCTCATGGAAGATCCCTCGGGCTGGTGGAAGGGCCGGCTTCACGGCCAG GAGGGCCTTTTCCCAGGAAACTACGTGGAGAAGATCTGAGCTGGGCCCTGGGATACTGCCTTCTCTTTCG CCCGCCTATCTGCCTGCCGGCCTGGTGGGGAGCCAGGCCCTGCCAATGAAAGCCTCGTTTACCTGGGCTG CAATAGCCTAAAAGTCCAATCCTTTGGCCTCCAGTCCTTGCCCAGGCCCTGGGTCACCAGGTCACTGGTG CAGCCCCCGCCCCTGGGCCCTGGTTTTCCTCCAACATCACACCTGCTGCCCATTGTCCAAAACTGTGTGT GTCAAAGGGGACTAACAGCAGAATTTACCTCCCAACTGCCATGTGATTAAGAAATGGGTCTTGAGTCCTG TGCTGTTGGCAAAGTTCCAGGCACAGTTGGGGAGGGGGGGCCGGAATCCGC FASTA File Format This is how genomic information is stored in the computer world.

  8. Single Nucleotide Polymorphisms (SNPs) An ontological perspective Mutation SNP Change in the base sequence of DNA Inherited or spontaneous Primary Cause of a Disease or Disorder Predisposes Carrier to Disease/Disorder Confers Disease Resistance to Carrier Effect of Base Change is Unknown

  9. Single Nucleotide Polymorphisms (SNPs) Typically, a SNP in a gene that encodes a drug metabolism enzyme will decrease the activity of the enzyme, thereby altering how well the body clears the drug. The Area Under the Curve (AUC) is a common representation of drug metabolism kinetics A normal (“mock”) patient’s AUC (solid line, lower left) following a standard warfarin oral dose shows the changes in drug plasma concentration over time. Warfarin is metabolized to 7-hydroxywarfarin by the oxidative metabolism enzyme 2C9, which is primary mechanism for warfarin clearance. There are two variant alleles that have a reduced capability for metabolizing warfarin, with 11% and 7% frequency in the Caucasian population for variants CYP2C9*2 and CYP2C9*3, respectively. Patients who are homozygous for these variant alleles (i.e. patients have two variant copies of the 2C9 gene) experience a 65% decrease in drug clearance rate 29 (dotted line, lower left). Note that the presence of a variant allele leads to increased drug plasma concentrations above the minimum toxic concentration and markedly increases the risk of an adverse drug response.

  10. Single Nucleotide Polymorphisms (SNPs) There are examples of SNPs in CYP genes (genes that encode P450 enzymes) that: • SNPs in the gene’s promoter region can increase or decrease gene expression levels, thereby altering the total amount of P450 enzyme in the liver. • SNPs in the CYP gene that do NOT have any effect on clearance rates for a particular drug.

  11. Single Nucleotide Polymorphisms (SNPs) Discovering SNPs and linking these to altered metabolism effects. 3 cohorts of people are evaluated (normals, heterozygous, and homozygous for allelic variant), dosed with a known drug (substrate) in a classic pharmacokinetic study. Biotechnology: DNA sequencing of cohort of people (ethnicity is important). Molecular Biology methods are utilized to express the altered P450 in a non-clinical model. SNP in CYP gene is discovered (i.e. an altered DNA sequence is found). Effect of SNP on enzyme activity is studied (in the test tube). Note that this is only useful for non-synonymous SNPs. Effect of SNP is reported, and utilized as rationale for additional studies in other known substrates. In this case, this may involve DNA studies in a cohort of patients already taking the drug that are experiencing altered efficacy or toxicity profiles. New SNP population frequency is determined.

  12. Where do we get DNA sequence information? DNA Sequencing Methods -conversion of biological/bioanalytical data into sequence information NOTE: There are automated, high-throughput sequencing centers that COMPLETELY automate (robotics and information systems) DNA sequencing, preliminary identification and publishing.

  13. A G C T TTTGGTCCGGCTATTCCATGATGTGCTTTTTTT TTGGTCCGGCTATTCCATGATGTGCTTTTTTT TGGTCCGGCTATTCCATGATGTGCTTTTTTT GGTCCGGCTATTCCATGATGTGCTTTTTTT GTCCGGCTATTCCATGATGTGCTTTTTTT TCCGGCTATTCCATGATGTGCTTTTTTT CCGGCTATTCCATGATGTGCTTTTTTT CGGCTATTCCATGATGTGCTTTTTTT GGCTATTCCATGATGTGCTTTTTTT GCTATTCCATGATGTGCTTTTTTT CTATTCCATGATGTGCTTTTTTT TATTCCATGATGTGCTTTTTTT ATTCCATGATGTGCTTTTTTT dATP dCTP dTTP dGTP ddATP32 ddCTP32 ddTTP32 ddGTP32 + DNA Sequencing (old method) 5’-AAACCAGGCCGATAAGGTACTACACGAAAAAAA-3’ TTTTTTT AAACCAGGCCGATAAGGTACTACACGAAAAA | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Step 1. Extend complementary sequence using “free” nucleotides with limiting amounts of radioactive “terminating” nucleotides. Step 2. Run product out on a electrophoresis gel. Step 3. Place gel against radiographic film, develop.

  14. DNA Sequencing new method http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html

  15. DNA Sequencing – SNP Discovery IUPAC = International Union of Pure and Applied Chemistry

  16. DNA Sequencing can be used for the Detection of known SNPs, but other more efficient, cost-effective, high-throughput biotechnology methods have been developed (and continue to be developed).

  17. 5’ 3’ C A G T A AC G G T T G T C A T TG C C A A 3’ 5’ Basics of DNA Detection The Key to DNA Detection is “Sequence-Specific Affinity” “GC” content (base paring) generally dictates thermodynamics of complementary binding. Tm = Melting Temperature

  18. “TARGET” is the fluorescence labeled DNA derived from the patient. Basics of DNA Detection “PROBE” is DNA attached to a fixed position

  19. Basics of DNA Detection • Three Major Methods of SNP Detection: • RFLP • Hybridization • Single-Base Extension • These biotechnology assays concatenate (A) a DNA sample preparation step, and (B) an analytical-instrument detection step. • Keep in mind that these SNP assays are aimed at KNOWN SNPs, and are developed to determine if the patient’s DNA sample is one of three states: • Homozygous normal • Heterozygous (one normal, one altered base) • Homozygous abnormal (both bases are altered)

  20. Basics of DNA Detection 2 copies of every CYP gene …AGATGCTCGATAATGATCGCTA… …TCTACGAGCTATTACTAGCGAT… Homozygous (NORMAL) …AGATGCTCGATAATGATCGCTA… …TCTACGAGCTATTACTAGCGAT… …AGATGCTCGATAATGATCGCTA… …TCTACGAGCTATTACTAGCGAT… Heterozygous …AGATGCTCGAGAATGATCGCTA… …TCTACGAGCTCTTACTAGCGAT… …AGATGCTCGAGAATGATCGCTA… …TCTACGAGCTCTTACTAGCGAT… Homozygous (ABNORMAL) …AGATGCTCGAGAATGATCGCTA… …TCTACGAGCTCTTACTAGCGAT…

  21. We will use CYP2C9*3 (7% frequency in Caucasian population) for our examples… What does a population frequency of 7% mean? >gi|13699817|ref|NM_000771.2| Homo sapiens cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9), mRNA ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTCTGGAGACAGAGCT CTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTGGAAATATCCTACAGATAGGTAT TAAGGACATCAGCAAATCCTTAACCAATCTCTCAAAGGTCTATGGCCCGGTGTTCACTCTGTATTTTGGC CTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGT TTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGAATTGTTTTCAGCAATGG AAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGC ATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGCCTCACCCT GTGATCCCACTTTCATCCTGGGCTGTGCTCCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTT TGATTATAAAGATCAGCAATTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGC CCCTGGATCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAATTAC TTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCAAGAATCAATGGACAT GAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATGGAGAAGGAAAAGCACAACCAACCATCT GAATTTACTATTGAAAGCTTGGAAAACACTGCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCA CAACCCTGAGATATGCTCTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGAT TGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCT GTGGTGCACGAGGTCCAGAGGTACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACA TTAAATTCAGAAACTATCTCATTCCCAAGGGCACAACCATATTAATTTCCCTGACTTCTGTGCTACATGA CAACAAAGAATTTCCCAACCCAGAGATGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAG AAAAGTAAATACTTCATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGG AGCTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAACCT TGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCATTCCTGTC TGAAGAAGAGCAGATGGCCTGGCTGCTGCTGTGCAGTCCCTGCAGCTCTCTTTCCTCTGGGGCATTATCC ATCTTTGCACTATCTGTAATGCCTTTTCTCACCTGTCATCTCACATTTTCCCTTCCCTGAAGATCTAGTG AACATTCGACCTCCATTACGGAGAGTTTCCTATGTTTCACTGTGCAAATATATCTGCTATTCTCCATACT CTGTAACAGTTGCATTGACTGTCACATAATGCTCATACTTATCTAATGTAGAGTATTAATATGTTATTAT TAAATAGAGAAATATGATTTGTGTATTATAATTCAAAGGCATTTCTTTTCTGCATGATCTAAATAAAAAG CATTATTATTTGCTG How many people (out of 1,000) would be heterozygous for CYP2C9*3? 70 How many people (out of 1,000) would be homozygous for CYP2C9*3? 5 How many people (out of 1,000) would be at risk for decreased CYP2C9 activity (*2 = 11%; *3 =7%)?

  22. Nonsynonymous mutations in CYP2C9 with functional effects Non-synonymous mutations with functional activity are listed. Those that functional activity has not been examined were not listed.

  23. Missense mutations with functional effects mapped in the crystal structure of human CYP2C9 protein bound with warfarin (PDB: 10G5). S-warfarin and heme are shown in the skeleton model with pink and red, respectively. Amino acid residues are shown in the sphere mode with colors.

  24. Biotechnologies - PCR Essentially all SNP detection methods utilize PCR (Polymerase Chain Reaction) as a “sample preparation” step to DRAMATICALLY INCREASE or AMPLIFY the small DNA region under investigation. PCR is by far the most common DNA molecular biology technique utilized, and is used for gene cloning, gene sequencing, most DNA analysis methods, BUT can ONLY be used in known genomic regions and models (i.e. the DNA sequence under investigation must have already been sequenced to utilize PCR).

  25. PCR Concept: Amplification of a relatively short piece of DNA for manipulation or sequencing. Driving phenomena of PCR: Heating and Cooling Heating: Double-stranded DNA “comes apart” when heated to near boiling. This is also called “denaturing” or “melting”. Cooling: Complementary DNA “comes together” when cooled. This is also called “renaturing”, “annealing” or “hybridizing”. Double-Stranded DNA COOLING HEATING Single-Stranded DNA

  26. Molecular Basis of PCR: Polymerase Activity A Polymerase is an enzyme that synthesizes DNA. 1) DNA can ONLY be synthesized using the complementary strand! 2) Polymerases synthesize DNA in the 5’ 3’ direction! 5’-GTCGATGTCTGATCAATTGGGCTGATCATGTCGATGATGCTAGAAT-3’ 3’CTACGATCTTA-5’ 5’-GTCGATGTCTGATCAATTGGGCTGATCATGTCGATGATGCTAGAAT-3’ ACTAGTACAGCTACTACGATCTTA-5’

  27. PCR uses the following reagents to AMPLIFY sections of DNA… • DNA template • Polymerase • Free Nucleotides (which are incorporated during DNA synthesis) • PCR Primers • Primers are two short pieces of DNA (each with a unique sequence) that are complementary to the two different strands of the DNA template. • In line diagrams, the primers are designated as arrows, where the arrows point in the direction of 3’ DNA synthesis.

  28. This section of the DNA template will be amplified. Double-Stranded DNA HEATING 3’ 5’ 5’ 3’ PCR Primers Single-Stranded DNA

  29. Double-Stranded DNA 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ HEAT (95ºC, 30 seconds) Single-Stranded DNA 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ COOL (60ºC, 30 seconds) PCR Primer Annealing 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCCTCCCCCACCGACCCCA-5’ 5’-GGATGGAACACTGGGGGGA-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’

  30. HEAT (72ºC, 30 seconds) Polymerase Elongation 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ CTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGA 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ DNA Synthesis after 1 “cycle” of PCR = 1 double stranded DNA is now 2 “copies” 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’

  31. 95ºC 30 Sec. 95ºC 30 Sec. 95ºC 30 Sec. 95ºC 30 Sec. 72ºC 30 Sec. 72ºC 30 Sec. 72ºC 30 Sec. 72ºC 30 Sec. 60ºC 30 Sec. 60ºC 30 Sec. 60ºC 30 Sec. 60ºC 30 Sec. “THERMOCYCLING” • Denaturing Step • Primer Annealing Step • Elongation Step 95ºC 30 Sec. 72ºC 30 Sec. Temperature 60ºC 30 Sec. Time

  32. Most PCR applications use 30 cycles (230 = 1.07 billion), representing an amplification of about 1 billion fold.

  33. Basics of DNA Detection • Three Major Methods of SNP Detection: • RFLP • Hybridization • Single-Base Extension • These biotechnology assays concatenate (A) a DNA sample preparation step, and (B) an analytical-instrument detection step. • Keep in mind that these SNP assays are aimed at KNOWN SNPs, and are developed to determine if the patient’s DNA sample is one of three states: • Homozygous normal • Heterozygous (one normal, one altered base) • Homozygous abnormal (both bases are altered)

  34. Biotechnologies - RFLP Restriction Fragment Length Polymorphism (RFLP, or sometimes called PCR-RFLP) is used to assay DNA sequences arising from their differing nucleotide sequences. 1) The DNA region that harbors the known SNP is amplified using PCR. 2) The PCR product (short double-stranded DNA) is treated (digested or cut) with a restriction enzyme, which cuts DNA at specific sequence sites. 3) The results of the restriction enzyme digestion is analyzed to determine the number and/or size of the resulting DNA strands. 2 Restriction Enzyme Digestion 1

  35. Biotechnologies - RFLP Using CYP2C9*3 (7% frequency in Caucasian population)… >gi|13699817|ref|NM_000771.2| Homo sapiens cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9), mRNA ATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCACTCTGGAGACAGAGCT CTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTGGAAATATCCTACAGATAGGTAT TAAGGACATCAGCAAATCCTTAACCAATCTCTCAAAGGTCTATGGCCCGGTGTTCACTCTGTATTTTGGC CTGAAACCCATAGTGGTGCTGCATGGATATGAAGCAGTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGT TTTCTGGAAGAGGCATTTTCCCACTGGCTGAAAGAGCTAACAGAGGATTTGGAATTGTTTTCAGCAATGG AAAGAAATGGAAGGAGATCCGGCGTTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGC ATTGAGGACCGTGTTCAAGAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGCCTCACCCT GTGATCCCACTTTCATCCTGGGCTGTGCTCCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTT TGATTATAAAGATCAGCAATTTCTTAACTTAATGGAAAAGTTGAATGAAAACATCAAGATTTTGAGCAGC CCCTGGATCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACAAATTAC TTAAAAACGTTGCTTTTATGAAAAGTTATATTTTGGAAAAAGTAAAAGAACACCAAGAATCAATGGACAT GAACAACCCTCAGGACTTTATTGATTGCTTCCTGATGAAAATGGAGAAGGAAAAGCACAACCAACCATCT GAATTTACTATTGAAAGCTTGGAAAACACTGCAGTTGACTTGTTTGGAGCTGGGACAGAGACGACAAGCA CAACCCTGAGATATGCTCTCCTTCTCCTGCTGAAGCACCCAGAGGTCACAGCTAAAGTCCAGGAAGAGAT TGAACGTGTGATTGGCAGAAACCGGAGCCCCTGCATGCAAGACAGGAGCCACATGCCCTACACAGATGCT GTGGTGCACGAGGTCCAGAGGTACATTGACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACA TTAAATTCAGAAACTATCTCATTCCCAAGGGCACAACCATATTAATTTCCCTGACTTCTGTGCTACATGA CAACAAAGAATTTCCCAACCCAGAGATGTTTGACCCTCATCACTTTCTGGATGAAGGTGGCAATTTTAAG AAAAGTAAATACTTCATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGG AGCTGTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAACCT TGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCATTCCTGTC TGAAGAAGAGCAGATGGCCTGGCTGCTGCTGTGCAGTCCCTGCAGCTCTCTTTCCTCTGGGGCATTATCC ATCTTTGCACTATCTGTAATGCCTTTTCTCACCTGTCATCTCACATTTTCCCTTCCCTGAAGATCTAGTG AACATTCGACCTCCATTACGGAGAGTTTCCTATGTTTCACTGTGCAAATATATCTGCTATTCTCCATACT CTGTAACAGTTGCATTGACTGTCACATAATGCTCATACTTATCTAATGTAGAGTATTAATATGTTATTAT TAAATAGAGAAATATGATTTGTGTATTATAATTCAAAGGCATTTCTTTTCTGCATGATCTAAATAAAAAG CATTATTATTTGCTG

  36. Biotechnologies - RFLP CYP2C9*1 GAGGTCCAGAGGTACATTGACCTTCTCCCCAC CYP2C9*3 GAGGTCCAGAGGTACCTTGACCTTCTCCCCAC Restriction Enzyme: Kpn I, which cuts at GGTACC

  37. Biotechnologies - RFLP PCR product = 105 base pairs, which spans the variant site. After KpnI digestions… 105 bp # of DNA Fragments 1 3 2 CYP2C9*1/*1 CYP2C9*1/*3 + 20 bp 85 bp CYP2C9*3/*3 20 bp 85 bp

  38. Biotechnologies - Hybridization In a hybridization-based SNP assay, the difference in DNA sequence is sufficient to disrupt “natural” double-stranded re-naturing / annealing / hybridization. This is accomplished by using relatively short DNA “capture probes”. In long strands of DNA, a single mismatched base pair is NOT sufficient to disrupt the formation of a double-stranded DNA “hybrid”. …TAGTCGCTAGATGATCG… …ATCAGCGAGCTACTAGC… >30 bp Note: This is NOT a SNP!!!, it is just an example of double-stranded DNA with a mismatched base pair!!!

  39. This section of the DNA template will be amplified. Biotechnologies – Hybridization DNA Microarray Technology • PCR used to generate short DNA strand that harbors the variant position. • PCR uses a “primer” with a fluorescent “tag” for detection. • PCR products are “hybridized” to the microarray surface, then analyzed. 3’ 5’ 5’ 3’ PCR Primers

  40. Biotechnologies – Hybridization DNA Microarray Technology SNP location Fluoro-PCR product Microarray = 1”x3” glass slide These 2 “spots” contain a different short DNA strand that is “complementary” to CYP2C9*1 or CYP2C9*2

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