Genomics & Biotechnology

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

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

Genomics Review

DNA is Information Storage

“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.

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.

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

>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.

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


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.


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.


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.

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.

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)

http://bioweb.uwlax.edu/GenWeb/Molecular/Seq_Anal/seq_anal.htm

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html

DNA Sequencing new method

http://bioweb.uwlax.edu/GenWeb/Molecular/Seq_Anal/seq_anal.htm

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).

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

“PROBE” is DNA attached to a fixed position

“TARGET” is the fluorescence labeledDNA derived from the patient.



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)


…AGATGCTCGATAATGATCGCTA……TCTACGAGCTATTACTAGCGAT…


Homozygous (NORMAL)

Heterozygous

Homozygous (ABNORMAL)


…AGATGCTCGAGAATGATCGCTA……TCTACGAGCTCTTACTAGCGAT…



2 copies of every CYP gene

>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%)?

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.

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”.

HEATING

COOLING

Double-Stranded DNA

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) 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.

HEATING

Double-Stranded DNA

Single-Stranded DNA

5’

3’

3’

5’PCR Primers

This section of the DNA templatewill be amplified.

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

5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ CTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’

5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGA3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’

Polymerase Elongation

HEAT (72ºC, 30 seconds)



DNA Synthesis after 1 “cycle” of PCR = 1 double stranded DNA is now 2 “copies”

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”

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


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)

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

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

GAGGTCCAGAGGTACATTGACCTTCTCCCCAC


GAGGTCCAGAGGTACCTTGACCTTCTCCCCAC

CYP2C9*1

CYP2C9*3

Restriction Enzyme: Kpn I, which cuts at GGTACC


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

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!!!

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.

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

Documents

Genomics & Biotechnology