1. Taqman Technology and Its Application to Epidemiology
EPI 243, April 21, 2009
Taqman Technology and Its Application to Epidemiology
Yuko You, M.S., Ph.D.

2. Current Techniques for SNP
PCR-RFLP
Taqman
Other technologies
Sequencing
Microarray (DNA chips)
SNPlex
Illumina SNP panel

3. DNA polymorphisms

4. 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)

5. 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”.

6. Common types of polymorphisms
SNP: Single Nucleotide Polymorphism or point mutation
Transition (A↔G or C↔T)
Transversion (less common)
Insertion/Deletion
Multiple Alleles
Microsatellite – variable number of tandom repeats
A T
C G
Transition: Purine to purine, pyrimidines to pyrimidines

7. 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

8. 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)

9. SNP Naming Restriction site Location rs number Substitutions Deletions
ALDH2-MboII
Location
TNF-α -308
rs number
Rs (dbSNP maps each submitted SNP assay to the genome and assigns a RefSNP accession ID (rs number) to each submitted SNP assay )
Rs =TNF-α -308
Substitutions
EX3+76A>C denotes that at nucleotide 76 of exon 3, an A is changed to a C
G487A
Deletions
EX5+76_78delACT denotes an ACT deletion from nucleotides 76 to 78 of exon 5
Insertions
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

10. SNP Naming For example: TNF-α
Ex3+19C>T (nucleotide 19 of exon 3, an C is changed to a T) rs
Pro84Leu
P84L

11. 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

12. 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

13. Haplotype
From Hapmap

14. Haplotypes AA AG GG CC A A C C A G G G C C CT C T A G A G C T or T C5’ 3’
Example for 2 SNPs haplotype combination
SNP1
SNP2
SNP1 AA AG GG CC
A A
C C
A G
G G
C C CT
C T
A G A G
C T or T C
C T TT
T T
T T
SNP2

15. PCR-RFLP

16. 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 .

17. How PCR is done ? Primers ---->
At 72 degree, the polymerase works best. As a result, the attraction, created by the hydrogen bonds, of the primers to the template is stronger than the forces breaking these attractions. The upshot is that bases complementary to the template are coupled to the primer

18. How PCR is done ?

19. The elements for PCR-RFLP
PCR reaction mix
DNA
PCR program
RFLP
Agarose gel electrophoresis
Genotype scoring

20. 1. PCR Reaction Mix

21. 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.

22. 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.

23. 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

24. 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
1 min
Annealing
55C
An annealing time of seconds was sufficient for all primer pairs tested so far. The annealing temperature can be chosen based on the melting temperature of the primers
Extension
72C
Last extension
Supposedly to help finish the elongation of many or most PCR products initiated during the last cycle
Storage
4C 

25. Typical PCR program x30 cycles 95C 72C 10min 1min 55C 7min 4C 
Depend on primers design

26. 5’-GTTTGGTTCTCTTCAGCGTGGAG-3’ 5-CATGAACCCTGGCAGGGTCTAAG-3’
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’
5-CATGAACCCTGGCAGGGTCTAAG-3’
Annealing time calculate
Tm = 4(G + C) + 2(A + T)°C
= 66°C
2 repeat=155
3 repeat=187
= 62°C

27. Primitive PCR machine

28. 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
Source:

29. List of Restriction Enzyme

30. PCR-RFLP
How a restriction enzyme work?
i.e. EcoRI

31. PCR-RFLP http://tools.neb.com/NEBcutter2/index.php
SNPicker: a graphical tool for primer picking in designing mutagenic endonuclease restriction assays.
Niu T,
Hu Z.

32. PCR-RFLP SNPicker Developed by Dr. Tianhua Niu and Dr. Zhenjun Hu
SNPicker: a graphical tool for primer picking in designing mutagenic endonuclease restriction assays.
Niu T,
Bioinformatics Nov 22;20(17): Epub 2004 Jun 16

33. 5. Agarose Gel Electrophoresis

34. 5. Agarose Gel Electrophoresis

35. 5. Agarose Gel Electrophoresis

36. 5. Agarose Gel Electrophoresis

37. 6. Genotype scoring Sample results of PCR-RFLP (CAPN10)12 22 11
1 = major allele
2 = minor allele

38. Taqman SNP assay

39. 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.

40. 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.

41. Taqman SNP assay A substantial increase in….. Indicates……
Each TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites.
When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Förster-type energy transfer (Förster, 1948; Lakowicz, 1983).
2.AmpliTaq Gold® DNA polymerase cleaves only probes that are hybridized to the target.
3.Cleavage separates the reporter dye from the quencher dye, which results in increased fluorescence by the reporter.
The increase in fluorescence signal occurs only if the amplified target sequence is complementary to the probe. Thus, the fluorescence signal generated by PCR amplification indicates which alleles are present in the sample.
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

42. Probe-Based Assay Chemistry

43. How is Fluorescence Data Collected?
Emission
Filter
Detector
Excitation
Filter
Light Source

44. How is Fluorescence Data Collected?

45. Standard Procedures for Taqman SNP assay

46. Taqman SNP assay The assays require only three components:
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.

47. Default Taqman SNP program
95C
92C
60C
10min
15sec
1min
x40 cycles

48. 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

49. Genotype scoring

50. Genotype scoring

51. Compare PCR-RFLP and Taqman
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

52. 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

53. Other Molecular Technologies
Direct Sequencing
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.
The key principle of the Sanger method was the use of dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators.

54. Other Molecular Technologies
Direct Sequencing (Continue)
The key principle of the Sanger method was the use of dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators.

55. Other Molecular Technologies
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

56. Other Molecular Technologies
SBE (Single base extension)
Allele specific primers designed
polymerase synthezises the primer on the template exact one base before the SNP
Pyrosequencing
short-read DNA sequencing and mutation/SNP analysis
SNPlex
Taqman Assay based (48-96 SNPs per reaction)
Illumina SNP panel
carried out in 384, 768, and 1536 plex formats using Illumina custom SNP panels or standard validated pre-manufactured panels
SNP analysis using Illumina Bead Station for GoldenGate Assays This platform is most suitable for researchers engaged in large scale association studies: whole genome linkage mapping panels and large scale fine mapping panels. The genotyping is carried out in 384, 768, and 1536 plex formats using Illumina custom SNP panels or standard validated pre-manufactured panels. The Illumina Bead Station Golden Gate assay operates using microbead technology assembled into 96 sample arrays with redundant bead types for increased confidence calls. The bead arrays are manufactured based on the custom SNP sets selected and configured onto the array surface. Incubation of the processed WGA or genomic DNA on the bead array allows hybridization to the appropriate probe on the bead and allows identification of a particular SNP. The system is also suitable for the use of the SNP genotyping data to analyze and visualize DNA copy number changes and to accurately characterize loss of heterozygosity (LOH). All custom SNP assay design is carried out using Illumina's SNP Knowledge Resource, which consists of a large SNP database and expert support service. This resource provides access to more than 1,000,000 high-confidence, mapped, and annotated SNP markers and to validated SNP assays across the human genome. Standard Validated pre-manufactured panels for Illumina GoldenGate Genotyping Assay
1. Human Cancer SNP Panel contains 1429 SNPs derived from 407 genes thought to be involved in cancer. The panel content was selected from the National Cancer Institute’s Cancer Genome Anatomy Project SNP500 Cancer Database Human Linkage Panel SNP markers (5,861) are distributed on every chromosome with an average gap of 482 Kb and 0.64 cM 3. Human Major Histocompatibility Panel Set consists of two oligonucleotide assay pools, the MHC Mapping Panel and the MHC Exon-Centric Panel. These two panels can be used independently or combined for more comprehensive coverage. The 2,360-loci set has the following attributes:
Average 2 kb between each SNP
93% loci, <5 kb spacing
98% loci, <10 kb spacing, in addition to even spacing across the MHC region.
4. Human DNA Test Panel includes 360 highly validated single nucleotide polymorphism (SNP) assays distributed across the genome with all chromosomes represented, including both X and Y for gender verification 5. The Mouse Low Density (LD) Linkage Panel consists of 377 loci, optimized for application to N2 and F2 mouse genetics crosses. These crosses can be used for mapping quantitative trait loci. The Mouse Medium Density (MD) Linkage Panel consists of 1,449 loci, optimized for various mapping applications including characterization of transgenic, congenic and knockout animals, and genetic mapping in advanced intercross mouse lines.
In the case of Illumina GoldenGate genotyping we recommend that at least 10% of samples be duplicated within the samples to act as QC, as no further QC of samples will be carried out in the lab.

57. 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

58. Multiplexing vs. Pooling
Many genotypes from one reaction carried out in one tube
Pooling
Reactions carried out separately, analyzed together