Transcript slides

EPI 243, April 21, 2009
Taqman Technology and Its
Application to Epidemiology
Yuko You, M.S., Ph.D.
Current Techniques for SNP



PCR-RFLP
Taqman
Other technologies
Sequencing
 Microarray (DNA chips)
 SNPlex
 Illumina SNP panel

DNA polymorphisms
DNA polymorphisms

More than 99% of nucleotides in DNA are the same in all
humans. The DNA loci that vary from person to person are said
to be “Polymorhic”, and the alternate sequence of the same
locus are called Allele.

Technically, the term “Polymorphisms” is generally restricted to
those variations that are relatively common (> 1% of
individuals)
DNA polymorphisms

Common variant allele usually are named “Wild-type” or
“Normal” allele, while rare or less common variant allele
are named “Mutant” or “Variant" allele.

Person with two copies of the same allele are
“Homozygous” and have different alleles on the two
chromosomes are “Heterozygous”.
Common types of
polymorphisms

SNP: Single Nucleotide Polymorphism or point
mutation
A
T
Transition (A↔G or C↔T)
 Transversion (less common)
 Insertion/Deletion
 Multiple Alleles


C
Microsatellite – variable number of tandom
repeats
G
SNP

SNPs make up 90% of all human genetic
variations

SNPs with a minor allele frequency of ≥ 1%
occur every 100 to 300 bases along the human
genome

On average, where two of every three SNPs
substitute C with T
SNP

Results from SNP
Synonymous
 Nonsynomous

Missense (change one amino acid
to another)
 Nonsense(changing an amino acid
to stop)
 Frameshift (results from
insertion/deletion)

SNP Naming

Restriction site


Location




EX5+76_78delACT denotes an ACT deletion from nucleotides 76 to 78 of exon 5
Insertions


EX3+76A>C denotes that at nucleotide 76 of exon 3, an A is changed to a C
G487A
Deletions


Rs (dbSNP maps each submitted SNP assay to the genome and assigns a RefSNP accession
ID (rs number) to each submitted SNP assay )
Rs1800629=TNF-α -308
Substitutions


TNF-α -308
rs number


ALDH2-MboII
76_77insT denotes that a T was inserted between nucleotides 76 and 77
Amino acid change

Pro187Ser denotes that SNP causes amino acid changes from Proline to Serine at amino acid
187
SNP Naming

For example: TNF-α
Ex3+19C>T (nucleotide 19 of exon 3, an C is
changed to a T)
 rs4645843
 Pro84Leu
 P84L

SNP Naming

Allele naming
By the nucleotide: AA, AC, CC
 By amino acid: Pro/Pro, Pro/Ser, Ser/Ser
 By default:

11, 12, 22 (1 as wildtype, 2 as variant)
 Or 00, 01, 11 (0 as wildtype, 1 as variant)


By Minor allele frequency count: 0, 1, 2
Haplotypes

A combination of alleles at multiple linked loci that are
transmitted together.

Haplotype may refer to as few as two loci or to an entire
chromosome depending on the number of
recombination events that have occurred between a
given set of loci.

The group of alleles of linked genes contributed by
either parent; the haploid genetic constitution
contributed by either parent
Haplotype
Haplotypes
Example for 2 SNPs haplotype combination
3’
5’
SNP1
SNP2
SNP1
AA
SNP2
CC
CT
TT
A
C
A
C
A
T
A
C
A
T
A
T
AG
A
C
A
C
A
T
G
C
G
T
G
T
or
A G
T C
GG
G
C
G
C
G
T
G
C
G
T
G
T
PCR-RFLP
What is PCR?

Polymerase Chain Reaction

A laboratory technique that can amplify the
amount of DNA from a tiny sample to a
large amount within just a few hours.

Applications for basic science,
epidemiology, evolution, linkage analysis,
forensics, anthropology
How PCR is done ?
Primers ---->
How PCR is done ?
The elements for PCR-RFLP
1.
2.
3.
4.
5.
6.
PCR reaction mix
DNA
PCR program
RFLP
Agarose gel electrophoresis
Genotype scoring
1. PCR Reaction Mix
1. PCR Reaction Mix
Component
CONTENT
FUNCTION
Water
H2O
Adjusted PCR reaction to appropriate concentration
PCR buffer
KCl, Tris and MgCl2
For their efficiency in supporting the activity of the
Taq polymerase.
dNTP
dATP, dTTP, dCTP, dGTP
deoxynucleotides
Provide both the energy and nucleosides for the
synthesis of DNA
DMSO
dimethyl sulphoxide
Improve amplification efficiency
Primers
Short pieces of DNA
Bind to DNA template allowing Taq DNA polymerase
enzyme to initiate incorporation of the
deoxynucleotides.
Taq
A heat stable enzyme that adds the deoxynucleotides to
the DNA template
DNA
The DNA template which will be amplified by the
PCR reaction.
Primers

Primers are short, artificial DNA strands — often not more
than 50 and usually only 18 to 25 base pairs long — that are
complementary to the beginning or the end of the DNA
fragment to be amplified.

They anneal by adhering to the DNA template at these
starting and ending points, where the DNA polymerase binds
and begins the synthesis of the new DNA strand.
Taq Polymerase – “Taq”



Taq polymerase is a thermostable DNA polymerase
named after the thermopilic bacterium “Thermus
aquaticus “ which lives in hot spring.
an enzyme able to withstand the protein-denaturing
conditions (high temperature) required during PCR
Taq's temperature optimum for activity is 75-80°C, with
a half-life of 9 minutes at 97.5°C, and can replicate a
1000 base pair strand of DNA in less than 10 seconds
at 72°C
3. PCR program (example)
Cycle
Temp
Time
NOTE
First
denaturing
94C
5 min
Many researchers use a 2-5 minutes first denaturing step
before the actual cycling starts. This is supposed to
help denaturing the target DNA better (especially the
hard to denature templates).
Denaturing
94C
1 min
Annealing
55C
1 min
Extension
72C
1 min
Last extension
72C
5 min
Storage
4C

An annealing time of 30-60 seconds was sufficient for all
primer pairs tested so far. The annealing temperature
can be chosen based on the melting temperature of
the primers
Supposedly to help finish the elongation of many or most
PCR products initiated during the last cycle
Typical PCR program
x30 cycles
95C
95C
10min
1min
72C
55C
1min
72C
7min
1min
4C

Depend on primers design
Example: CAPN10
25741 acgtgctctg cctgccgaag tgaggaggct gggcacggtg cctgggttcc ccctgcccag
25801 gcccagtttg gttctcttca gcgtggagag atgattctgt cccaggagcc gggaggaggg
25861 tgatgattct gtcccaggag ctgggaggag ggtgggcttg tgggaggggc tggctctgtc
25921 tgtggccgta gctgctgctt agaccctgcc agggttcatg aggccaccgt ggcgggaggc
25981 cagcgaggag ccgtgtccca cagctgatgc ctggtgtttt ctcactagag aggctgctct
26041 gccatacgcg ggcgctgcct ggggcctggg tcaagggcca gtcagcagga ggctgccgga
5’-GTTTGGTTCTCTTCAGCGTGGAG-3’ = 66°C
5-CATGAACCCTGGCAGGGTCTAAG-3’

Annealing time calculate
Tm = 4(G + C) + 2(A + T)°C
= 62°C
Primitive PCR machine
4. PCR-RFLP

Restriction fragment length polymorphism

The purpose is to detection of point mutations after the
genomic sequences are amplified by the PCR

A restriction enzyme (or restriction endonuclease) is an
enzyme that cuts double-stranded DNA. The recognition sites
are usually 4 to 6 base pairs in length

Use gel electrophoresis easily identifies the mutations, since
mutant allele generate smaller DNA fragments
List of Restriction Enzyme
PCR-RFLP

How a restriction enzyme work?

i.e. EcoRI
PCR-RFLP

http://tools.neb.com/NEBcutter2/index.php
PCR-RFLP

SNPicker

Developed by Dr. Tianhua Niu and Dr. Zhenjun Hu
5. Agarose Gel Electrophoresis
5. Agarose Gel Electrophoresis
5. Agarose Gel Electrophoresis
5. Agarose Gel Electrophoresis
6. Genotype scoring

Sample results of PCR-RFLP (CAPN10)
12
22
12
22
22
22
22
12
11
22
12
22
11
11
12
12
12
12
11
12
12
22
22
12
22
12
12
11
12
11
12
22
11
1 = major allele
2 = minor allele
11
Taqman SNP assay
Taqman SNP assay

TaqMan SNP Genotyping Assays provide
optimized assays for genotyping single
nucleotide polymorphisms (SNPs).

The products use the 5´ nuclease assay for
amplifying and detecting specific SNP alleles in
purified genomic DNA samples.
Taqman SNP assay

The TaqMan SNP probe contains a reporter dye at the
5´ end of the probe and a quencher dye at the 3´ end
of the probe.

During the reaction, cleavage of the probe separates
the reporter dye and the quencher dye, which results in
increased fluorescence of the reporter.

Accumulation of PCR products is detected directly by
monitoring the increase in fluorescence of the reporter
dye.
Taqman SNP assay
A substantial increase in…..
Indicates……
VIC dye fluorescence only
Homozygosity for Allele 1
FAM dye fluorescence only
Homozygosity for Allele 2
Both fluorescence signals
Allele 1-Allele 2 Heterozygosity
Probe-Based Assay Chemistry
How is Fluorescence Data Collected?
Emission
Filter
Excitation
Filter
Light Source
Detector
How is Fluorescence Data Collected?
Standard Procedures for Taqman SNP assay
Taqman SNP assay

The assays require only three components:
1.
2.
3.

1 to 20 ng of purified genomic DNA template
SNP Genotyping Assay Mix (specific for each
polymorphism)
TaqMan Universal PCR Master Mix
The assays require only one amplification step
and an endpoint reading to obtain results.
Default Taqman SNP program
x40 cycles
95C
10min
92C
15sec
60C
1min
SNP Genotyping Assay Mix

Sequence-specific forward and reverse primers to
amplify the SNP of interest

Two Taqman Probes



One probe labeled with VIC® dye detects the Allele 1 sequence
One probe labeled with FAM™ dye detects the Allele 2 sequence
Assay designed specifically customized to fit the default
PCR program
Genotype scoring
Genotype scoring
Compare PCR-RFLP and Taqman
PCR-RFLP
Taqman SNP assay
Advantage
Inexpensive
for small setting studies
Flexible for most kind of SNP genotyping
Easy
to perform
Suitable for high throughput genotyping
Need less information about target
sequence
Less prone to human error
Faster, more efficient for SNP genotyping
Disadvantage
Need
information about target sequence
Not suitable for high throughput
genotyping
Prone to human error
Not
possible for all SNPs assays
Higher expenses for equipment
maintenance
Limitations of Taqman Assay





Not applicable for multi-allele SNPs
No deletion
No insertion
No Microsatellites
May not work if too many SNPs closed to the
targeting site
Other Molecular Technologies
Direct Sequencing
1.


DNA sequencing is the process of
determining the nucleotide order of a
given DNA fragment
DNA fragments can be labeled by using
a radioactive or fluorescent tag on the
primer, in the new DNA strand with a
labeled dNTP, or with a labeled ddNTP.
Other Molecular Technologies
1.
Direct Sequencing (Continue)
Other Molecular Technologies
2.
Microarray Chips

Microarrays measure gene expression by taking advantage of the
process of hybridization. Hybridization allows researchers to test
whether two pieces of DNA are complementary.

Some form of DNA spotted on a chip (probes)

Density of spots important

One individual sample, many genotypes per assay
Other Molecular Technologies
3.
SBE (Single base extension)

Allele specific primers designed

polymerase synthezises the primer on the template exact one base
before the SNP
4.
Pyrosequencing

short-read DNA sequencing and mutation/SNP analysis
5.
SNPlex

Taqman Assay based (48-96 SNPs per reaction)
6.
Illumina SNP panel

carried out in 384, 768, and 1536 plex formats using Illumina custom
SNP panels or standard validated pre-manufactured panels
Adaptation of method

Many SNPs (>1000) few samples (< 600)

Array platform



Many genotypes per array
Few arrays per day
Few SNPs (< 100) many samples (> 600)

Liquid based platform



Multi-well plates provide flexibility
Robotics can increase throughput
Usually many samples/one SNP per plate

Not true in “multiplex” situations
Multiplexing vs. Pooling

Multiplex


Many genotypes from one reaction carried out in
one tube
Pooling

Reactions carried out separately, analyzed together