genomics & biotechnology michael d. kane, phd asst. professor, department of computer &...
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Genomics & Biotechnology
Michael D. Kane, PhD
Asst. Professor, Department of Computer & Information TechnologyLead Genomic Scientist, Bindley Bioscience Center
Purdue University
Adjunct Asst. Professor of PharmacologyOhio Northern University
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1. Genomics Review2. Single Nucleotide Polymorphisms (SNPs)3. Basics of DNA Detection4. SNP Discovery5. SNP Detection6. Biotechnologies7. Data Formats8. Genomic Data serving as Clinical Decision Support
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Genomics Review
DNA is Information Storage
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“Zipped Files”
Decompression
“Executable Files”
Genomics Review
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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.
Genomics Review
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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.
5’3’
3’5’
5’3’
3’5’
AGTCGTGATCTGCTAAATGTCTCGAAGTTCGATGCTAG||||||||||||||||||||||||||||||||||||||TCAGCACTAGACGATTTACAGAGCTTCAAGATACGATC
Courier font is preferred for writing sequence data since letter spacingis independent of character content.
Genomics Review
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>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
FASTAFileFormat
This is how genomic information is stored in the computer world.
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Single Nucleotide Polymorphisms (SNPs)
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
An ontological perspective
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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.
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Single Nucleotide Polymorphisms (SNPs)
There are examples of SNPs in CYP genes (genes that encode P450 enzymes) that:
1. 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.
2. SNPs in the CYP gene that do NOT have any effect on clearance rates for a particular drug.
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Single Nucleotide Polymorphisms (SNPs)
Discovering SNPs and linking these to altered metabolism effects.
Biotechnology: DNA sequencing
of cohort of people (ethnicity is important).
SNP in CYP gene is discovered (i.e. an altered DNA
sequence is found).
New SNP population
frequency is determined.
3 cohorts of people are evaluated (normals, heterozygous, and
homozygous for allelic variant), dosed with a
known drug (substrate) in a classic pharmacokinetic
study.
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.
Molecular Biology methods are
utilized to express the altered P450 in
a non-clinical model.
Effect of SNP on enzyme activity is
studied (in the test tube). Note that this is only useful for non-synonymous
SNPs.
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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.
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A G C T
5’-AAACCAGGCCGATAAGGTACTACACGAAAAAAA-3’
dATPdCTPdTTPdGTP
+ddATP32
ddCTP32
ddTTP32
ddGTP32 TTTGGTCCGGCTATTCCATGATGTGCTTTTTTTTTGGTCCGGCTATTCCATGATGTGCTTTTTTT
TGGTCCGGCTATTCCATGATGTGCTTTTTTTGGTCCGGCTATTCCATGATGTGCTTTTTTT
GTCCGGCTATTCCATGATGTGCTTTTTTTTCCGGCTATTCCATGATGTGCTTTTTTT
CCGGCTATTCCATGATGTGCTTTTTTTCGGCTATTCCATGATGTGCTTTTTTT
GGCTATTCCATGATGTGCTTTTTTTGCTATTCCATGATGTGCTTTTTTT
CTATTCCATGATGTGCTTTTTTTTATTCCATGATGTGCTTTTTTT
ATTCCATGATGTGCTTTTTTT
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.
TTTTTTT
AAACCAGGCCGATAAGGTACTACACGAAAAA | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
DNA Sequencing (old method)
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http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html
DNA Sequencing new method
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DNA Sequencing – SNP Discovery IUPAC code Meaning
A A
C C
G G
T T
M A or C
R A or G
W A or T
S C or G
Y C or T
K G or T
V A or C or G
H A or C or T
D A or G or T
B C or G or T
N G or A or T or C
IUPAC = International Union of Pure and Applied Chemistry
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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).
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The Key to DNA Detection is “Sequence-Specific Affinity”
CAGTAACGGTT
5’
3’
GTCATTGCCAA
5’
3’
“GC” content (base paring) generally dictates thermodynamics of complementary binding. Tm = Melting Temperature
Basics of DNA Detection
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“PROBE” is DNA attached to a fixed position
“TARGET” is the fluorescence labeledDNA derived from the patient.
Basics of DNA Detection
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Basics of DNA Detection
Three Major Methods of SNP Detection:
1) RFLP2) Hybridization3) 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:
i) Homozygous normalii) Heterozygous (one normal, one altered base)iii) Homozygous abnormal (both bases are altered)
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Basics of DNA Detection
…AGATGCTCGATAATGATCGCTA……TCTACGAGCTATTACTAGCGAT…
…AGATGCTCGATAATGATCGCTA……TCTACGAGCTATTACTAGCGAT…
Homozygous (NORMAL)
Heterozygous
Homozygous (ABNORMAL)
…AGATGCTCGATAATGATCGCTA……TCTACGAGCTATTACTAGCGAT…
…AGATGCTCGAGAATGATCGCTA……TCTACGAGCTCTTACTAGCGAT…
…AGATGCTCGAGAATGATCGCTA……TCTACGAGCTCTTACTAGCGAT…
…AGATGCTCGAGAATGATCGCTA……TCTACGAGCTCTTACTAGCGAT…
2 copies of every CYP gene
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>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
CYP Family
Allele Nucleotide Change
Enzyme Activity Change
Associated Drug Concentration
Change
1A2 CYP1A2*1C -3860 G>C Decreases Increases
2C9 CYP2C9*3A 1075 A>C Decreases Increases
3A4 CYP3A4*18A 878 T>C Increases Decreases
We will use CYP2C9*3 (7% frequency in Caucasian population) for our examples…
What does a population frequency of 7% mean?
How many people (out of 1,000) would be heterozygous for CYP2C9*3?
How many people (out of 1,000) would be homozygous for CYP2C9*3?
70
5
How many people (out of 1,000) would be at risk for decreased CYP2C9 activity (*2 = 11%;*3 =7%)?
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Nonsynonymous mutations in CYP2C9 with functional effects
Alleles Nucleotide change in cDNA
Amino acid change
Enzymatic activity
CYP2C9 * 2430C > T Arg144Cys Decrease: an approximately 50% decrease of the maximum rate of metabolism (Vmax)
and 30–50% lower turnover (kcat) of S-warfarin
CYP2C9 * 31075A > C Ile359Leu Decrease: a markedly higher Km and lower intrinsic clearance with an approximately
90% decrease of S-warfarin
CYP2C9 * 41076T > C Ile359Thr Decrease: 72–81% reduction of intrinsic clearance of diclofenac
CYP2C9 * 51080C > G Asp360Glu Decrease: intrinsic clearance of warfarin approximately 10% of wild type
CYP2C9 * 6del818A Frame shift Null
CYP2C9 * 8449G > A Arg150His Increase: more than two-fold increase in the intrinsic clearance of tolbutamide
CYP2C9 * 111003C > T Arg335Trp Decrease: a three-fold increase in the Km and more than a two-fold decrease in the
intrinsic clearance of tolbutamide
CYP2C9 * 121465C > T Pro489Ser Decrease: a modest decrease in the Vmax and the intrinsic clearance of tolbutamide
CYP2C9 * 13269T > C Leu90Pro Decrease: decreased activity toward all studied CYP2C9 substrates
CYP2C9 * 14374G > A Arg125His Decrease: 80–90% lower catalytic activity toward tolbutamide
CYP2C9 * 15485C > A Ser162X Null
CYP2C9 * 16895A > G Thr299Ala Decrease: 80–90% lower catalytic activity toward tolbutamide
CYP2C9 * 171144C > T Pro382Ser Decrease: modest 30 to 40% decreases in caltalytic activity toward tolbutamide
CYP2C9 * 191362G > C Gln454His Decrease: modest 30 to 40% decreases in caltalytic activity toward tolbutamide
Non-synonymous mutations with functional activity are listed. Those that functional activity has not been examined were not listed.
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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.
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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).
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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”.
HEATING
COOLING
Double-Stranded DNA
Single-Stranded DNA
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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’
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PCR uses the following reagents to AMPLIFY sections of DNA…
1) DNA template2) Polymerase3) Free Nucleotides (which are incorporated during DNA synthesis)4) 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.
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HEATING
Double-Stranded DNA
Single-Stranded DNA
5’
3’
3’
5’PCR Primers
This section of the DNA templatewill be amplified.
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5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
Double-Stranded DNA
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’
3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
HEAT (95ºC, 30 seconds)
Single-Stranded DNA
COOL (60ºC, 30 seconds)
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCCTCCCCCACCGACCCCA-5’
5’-GGATGGAACACTGGGGGGA-3’3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
PCR Primer Annealing
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5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ CTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGA3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
Polymerase Elongation
HEAT (72ºC, 30 seconds)
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’
DNA Synthesis after 1 “cycle” of PCR = 1 double stranded DNA is now 2 “copies”
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95ºC30 Sec.
60ºC30 Sec.
72ºC30 Sec.
1) Denaturing Step2) Primer Annealing Step3) Elongation Step
Time
Tem
pera
ture
95ºC30 Sec.
60ºC30 Sec.
72ºC30 Sec.
95ºC30 Sec.
60ºC30 Sec.
72ºC30 Sec.
95ºC30 Sec.
60ºC30 Sec.
72ºC30 Sec.
95ºC30 Sec.
60ºC30 Sec.
72ºC30 Sec.
“THERMOCYCLING”
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Most PCR applications use 30 cycles (230 = 1.07 billion), representing an amplification of about 1 billion fold.
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Basics of DNA Detection
Three Major Methods of SNP Detection:
1) RFLP2) Hybridization3) 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:
i) Homozygous normalii) Heterozygous (one normal, one altered base)iii) Homozygous abnormal (both bases are altered)
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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.
RestrictionEnzymeDigestion
1
2
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Using CYP2C9*3 (7% frequency in Caucasian population)…
Biotechnologies - RFLP
>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
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GAGGTCCAGAGGTACATTGACCTTCTCCCCAC
Biotechnologies - RFLP
GAGGTCCAGAGGTACCTTGACCTTCTCCCCAC
CYP2C9*1
CYP2C9*3
Restriction Enzyme: Kpn I, which cuts at GGTACC
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Biotechnologies - RFLP
CYP2C9*1/*1
PCR product = 105 base pairs, which spans the variant site.
After KpnI digestions…105 bp
CYP2C9*1/*3
CYP2C9*3/*3
# of DNAFragments
1
3
2
+
85 bp 20 bp
85 bp 20 bp
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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”.
>30 bp …TAGTCGCTAGATGATCG……ATCAGCGAGCTACTAGC…
Note: This is NOT a SNP!!!, it is just an example of double-stranded DNA with a mismatched base pair!!!
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Biotechnologies – Hybridization
DNA Microarray Technology
1) PCR used to generate short DNA strand that harbors the variant position.2) PCR uses a “primer” with a fluorescent “tag” for detection.3) PCR products are “hybridized” to the microarray surface, then analyzed.
5’
3’
3’
5’PCR Primers
This section of the DNA templatewill be amplified.
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Biotechnologies – Hybridization
DNA Microarray Technology
Fluoro-PCR product SNP location
Microarray = 1”x3” glass slide
These 2 “spots” contain a different short DNA strand that is “complementary” to CYP2C9*1 or CYP2C9*2