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
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Genomics Review
Single Nucleotide Polymorphisms (SNPs)
Basics of DNA Detection
SNP Discovery
SNP Detection
Biotechnologies
Data Formats
Genomic Data serving as Clinical Decision Support
Genomics Review
DNA is Information Storage
Genomics Review
“Zipped Files”
Decompression
“Executable Files”
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.
DNA is Double Stranded…
Genomics Review
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 spacing
is independent of character content.
FASTA
File
Format
This is how
genomic
information
is stored in
the
computer
world.
>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
Single Nucleotide Polymorphisms (SNPs)
Mutation
An ontological perspective
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
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.
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.
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.
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 nonsynonymous
SNPs.
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.
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.
DNA Sequencing (old method)
5’-AAACCAGGCCGATAAGGTACTACACGAAAAAAA-3’
TTTTTTT
dATP
dCTP
dTTP
dGTP
+
ddATP32
ddCTP32
ddTTP32
ddGTP32
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.
A G C T
AAACCAGGCCGATAAGGTACTACACGAAAAA
|||||||||||||||||||||||||||||||||||||||
TTTGGTCCGGCTATTCCATGATGTGCTTTTTTT
TTGGTCCGGCTATTCCATGATGTGCTTTTTTT
TGGTCCGGCTATTCCATGATGTGCTTTTTTT
GGTCCGGCTATTCCATGATGTGCTTTTTTT
GTCCGGCTATTCCATGATGTGCTTTTTTT
TCCGGCTATTCCATGATGTGCTTTTTTT
CCGGCTATTCCATGATGTGCTTTTTTT
CGGCTATTCCATGATGTGCTTTTTTT
GGCTATTCCATGATGTGCTTTTTTT
GCTATTCCATGATGTGCTTTTTTT
CTATTCCATGATGTGCTTTTTTT
TATTCCATGATGTGCTTTTTTT
ATTCCATGATGTGCTTTTTTT
DNA Sequencing new method
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html
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
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).
Basics of DNA Detection
The Key to DNA Detection is “Sequence-Specific Affinity”
5’ 3’
G
T
C
A
T
T
G
C
C
A
A
C
A
G
T
A
A
C
G
G
T
T
3’ 5’
“GC” content (base paring) generally dictates thermodynamics of complementary binding. Tm = Melting Temperature
Basics of DNA Detection
“TARGET” is the fluorescence labeled
DNA derived from the patient.
“PROBE” is DNA attached to a fixed position
Basics of DNA Detection
Three Major Methods of SNP Detection:
1) RFLP
2) Hybridization
3) 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 normal
ii) Heterozygous (one normal, one altered base)
iii) Homozygous abnormal (both bases are altered)
Basics of DNA Detection
2 copies of every CYP gene
…AGATGCTCGATAATGATCGCTA…
…TCTACGAGCTATTACTAGCGAT…
Homozygous (NORMAL)
Heterozygous
…AGATGCTCGATAATGATCGCTA…
…TCTACGAGCTATTACTAGCGAT…
…AGATGCTCGATAATGATCGCTA…
…TCTACGAGCTATTACTAGCGAT…
…AGATGCTCGAGAATGATCGCTA…
…TCTACGAGCTCTTACTAGCGAT…
Homozygous (ABNORMAL)
…AGATGCTCGAGAATGATCGCTA…
…TCTACGAGCTCTTACTAGCGAT…
…AGATGCTCGAGAATGATCGCTA…
…TCTACGAGCTCTTACTAGCGAT…
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?
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%)?
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
>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
Nonsynonymous mutations in CYP2C9 with functional effects
Alleles
Nucleotide
change in
cDNA
Amino acid
change
Enzymatic activity
CYP2C9
*2
430C > T
Arg144Cys
Decrease: an approximately 50% decrease of the maximum rate of metabolism (Vmax)
and 30–50% lower turnover (kcat) of S-warfarin
CYP2C9
*3
1075A > C
Ile359Leu
Decrease: a markedly higher Km and lower intrinsic clearance with an approximately
90% decrease of S-warfarin
CYP2C9
*4
1076T > C
Ile359Thr
Decrease: 72–81% reduction of intrinsic clearance of diclofenac
CYP2C9
*5
1080C > G
Asp360Glu
Decrease: intrinsic clearance of warfarin approximately 10% of wild type
CYP2C9
*6
del818A
Frame shift
Null
449G > A
Arg150His
Increase: more than two-fold increase in the intrinsic clearance of tolbutamide
CYP2C9
*8
CYP2C9
* 11
1003C > T
Arg335Trp
Decrease: a three-fold increase in the Km and more than a two-fold decrease in the
intrinsic clearance of tolbutamide
CYP2C9
* 12
1465C > T
Pro489Ser
Decrease: a modest decrease in the Vmax and the intrinsic clearance of tolbutamide
CYP2C9
* 13
269T > C
Leu90Pro
Decrease: decreased activity toward all studied CYP2C9 substrates
CYP2C9
* 14
374G > A
Arg125His
Decrease: 80–90% lower catalytic activity toward tolbutamide
CYP2C9
* 15
485C > A
Ser162X
Null
CYP2C9
* 16
895A > G
Thr299Ala
Decrease: 80–90% lower catalytic activity toward tolbutamide
CYP2C9
* 17
1144C > T
Pro382Ser
Decrease: modest 30 to 40% decreases in caltalytic activity toward tolbutamide
CYP2C9
* 19
1362G > 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.
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.
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).
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
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’
PCR uses the following reagents to AMPLIFY sections of DNA…
1)
2)
3)
4)
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.
Double-Stranded DNA
This section of the DNA template
will be amplified.
HEATING
3’
5’
PCR Primers
5’
3’
Single-Stranded DNA
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’
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’
95ºC
30 Sec.
Temperature
72ºC
30 Sec.
1) Denaturing Step
2) Primer Annealing Step
3) Elongation Step
60ºC
30 Sec.
Time
95ºC
30 Sec.
95ºC
30 Sec.
95ºC
30 Sec.
72ºC
30 Sec.
60ºC
30 Sec.
95ºC
30 Sec.
72ºC
30 Sec.
60ºC
30 Sec.
72ºC
30 Sec.
60ºC
30 Sec.
“THERMOCYCLING”
72ºC
30 Sec.
60ºC
30 Sec.
Most PCR applications use 30 cycles (230 = 1.07 billion),
representing an amplification of about 1 billion fold.
Basics of DNA Detection
Three Major Methods of SNP Detection:
1) RFLP
2) Hybridization
3) 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 normal
ii) Heterozygous (one normal, one altered base)
iii) Homozygous abnormal (both bases are altered)
Biotechnologies - RFLP
Restriction Fragment Length Polymorphism (RFLP, or sometimes called PCRRFLP) 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
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
Biotechnologies - RFLP
CYP2C9*1
GAGGTCCAGAGGTACATTGACCTTCTCCCCAC
CYP2C9*3
GAGGTCCAGAGGTACCTTGACCTTCTCCCCAC
Restriction Enzyme: Kpn I, which cuts at GGTACC
Biotechnologies - RFLP
PCR product = 105 base pairs, which spans the variant site.
After KpnI digestions…
105 bp
# of DNA
Fragments
1
CYP2C9*1/*1
CYP2C9*1/*3
3
+
85 bp
20 bp
2
CYP2C9*3/*3
85 bp
20 bp
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 doublestranded DNA with a mismatched
base pair!!!
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.
This section of the DNA template
will be amplified.
3’
5’
PCR Primers
5’
3’
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