西亚试剂：：HapMap assay-design protocols

 
Protocols for HapMap assay-design
Affymetrix platform (used by Broad)
Defined protocols:
LSID:  urn:LSID:affymetrix.hapmap.org:Protocol:affy_assay_design_1:1
Title: Genotyping using Affymetrix arrays
Description: Genome Complexity Reduction

Sample DNAs should not be highly degraded nor contain PCR inhibitors, such as high
concentrations of heme or chelating agents. For each individual assayed, 250 ng of
genomic DNA are digested separately with 10 U of XbaI or HindIII (New England
BioLabs) in volumes of 20 µL for 2 hours at 37 °C. Following heat inactivation at 70 °C
for 20 minutes, 0.25 µM of XbaI adaptor (5’-ATT ATG AGC ACG ACA GAC GCC
TGA TCT-3’ and 5’phosphate –CTA GAG ATC AGG CGT CTG TCG TGC TCA TAA-
3’)(Affymetrix), or HindIII adaptor (5’-ATT ATG AGC ACG ACA GAC GCC TGA
TCT-3’ and 5’phosphate –AGC TAG ATC AGG CGT CTG TCG TGC TCA TAA-3’)
(Affymetrix) are ligated to the digested DNAs with T4 DNA Ligase (New England
BioLabs) in 25 µL for 2 hours at 16 °C. The ligations are stopped by heating to 70 °C for
20 minutes, and then diluted 4- fold with water. For each ligation reaction, two to three
PCRs are run in order to generate > 40 µg of PCR products. Each PCR contains 10 µL of
the diluted ligation reactions (25 ng of starting DNA) in 100 µL volumes containing 1.0
µM of primer (5’-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3’), 0.30 mM
dNTPs, 1.0 mM MgSO4, 5 U Platinum® Pfx Polymerase (Invitrogen), PCR Enhancer
(Invitrogen) and Pfx Amplification Buffer (Invitrogen). 30 cycles of PCRs are run with
the following cycling program: 94 °C denaturation for 15 seconds, 60 °C annealing for
30 seconds, and 68 °C extension for 60 seconds. As a check, 3 µL of PCR products are
visualized on 2% TBE agarose gels to confirm the size range of amplicons. The PCR
products are purified over MinElute 96 UF PCR Purification plates (Qiagen), and
recovered in 40 µL of EB buffer (Qiagen). PCR yields are measured by absorbance
readings at 260 nm, and adjusted to a concentration of 40 µg per 45 µl. To allow
efficient hybridization to 25-mer oligonucleotide probes, the PCR products are
fragmented to < 100 bp with DNAse I. 0.20 U of DNAse I (Affymetrix) is added to 40
ug of purified PCR amplicons in a 55 µL volume containing Fragmentation Buffer
(Affymetrix) for 35 minutes at 37 °C, followed by heat inactivation at 95 °C for 15
minutes. Fragmentation products are visualized on 4% TBE agarose gels. The 3’ ends of
the fragmented amplicons are biotinlyated by adding 214 µM of a proprietary DNA
labeling reagent (Affymetrix) using Terminal Deoxynucleotidyl Transferase (Affymetrix)
in 70 µL volumes for 2 hours at 37 °C, followed by heat inactivation at 95 °C for 15
minutes.

Allele Specific Hybridization to Oligonucleotide Arrays

The fragmented and biotinylated PCR amplicons are combined with 11.5 µg/mL human
Cot-1 (Invitrogen) and 115 µg/mL herring sperm (Promega) DNAs. The DNAs are
added to a hybridization solution containing 2.69 M tetramethylamonium chloride
(TMACl), 5.77 mM EDTA, 56 mM MES, 5 % DMSO, 2.5 X Denhardt’s solution, and
0.0115% Tween-20 in a final volume of 260 µL. The hybridization solution was heated
to 95 °C for 10 minutes then placed on ice. After warming to 48 °C for 2 minutes, 200
µL of the hybridization solution is injected into cartridges housing the oligonucleotide
arrays (Affymetrix GeneChip® 100K Mapping Set: 50K Array Xba 240 and 50K Array
Hind 240). Hybridizations are carried out at 48 °C for 16 to 18 hours in a rotisserie
rotating at 60 rpm. Following the overnight hybridization, the arrays are washed with 6X
SSPE and 0.01% Tween-20 at 25 °C, then more stringently washed with 0.6X SSPE and
0.01% Tween-20 at 45 °C. Hybridization signals are generated in a three step signal
amplification process: 10µg/mL streptavidin R-phycoerythrin (SAPE) conjugate
(Molecular Probes) is added to the biotinylated targets hybridized to the oligonucleotide
probes, and washed with 6X SSPE and 0.01% Tween-20 at 25 °C; followed by the
addition of 5µg/mL biotinylated goat anti-streptavidin (Vector) to increase the effective
number of biotin molecules on the target; and finally SAPE is added once again and
washed extensively with 6X SSPE and 0.01% Tween-20 at 30 °C. The SAPE and
antibody were added to arrays in 6X SSPE, 1X Denhardt’s solution and 0.01% Tween-20
at 25 °C for 10 minutes each. Following the final wash, the arrays are kept in Holding
buffer (100mM MES, 1M [Na+], 0.01% Tween-20). The washing and staining
procedures are run on Affymetrix fluidics stations. Arrays are scanned using GCS3000
scanners with AutoLoaders (Affymetrix). Scan images are processed to get hybridization
signal intensity values using GCOS 2.0 software (Affymetrix). The DM genotype calling
algorithm is implemented in GenoTyping Tools (GTT) (Affymetrix) and GDAS 3.0
(Affymetrix) analysis software.

(from Hajime Matsuzaki, Shoulian Dong, Halina Loi, Xiaojun Di, Guoying Liu,Earl Hubbell, Jane Law, Tam Berntsen, Monica Chadha, Henry Hui, Geoffrey Yang, Giulia C Kennedy, Teresa A Webster, Simon Cawley, P Sean Walsh, Keith W Jones, Stephen P A Fodor & Rui Mei. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nature Methods  1, 109 - 111 (2004) . PubMed ID: 15782172)

BeadArray platform (used by Broad, Illumina, Sanger, McGill, Beijing, Shanghai/Taipei)
Defined protocols:
LSID:  urn:LSID:illumina.hapmap.org:Protocol:Golden_Gate_assay_design_1.0.0:1
Title: GoldenGateTM Assay Design Protocol
Description:
Candidate SNPs first undergo an informatic screen for suitability for assay design.  SNPs
can be excluded from assay design if they are located near or within palindromic sequences,
GT- or AT-rich regions or repeats similar to sequences elsewhere in the genome.
Three assay oligos are designed for each SNP locus that passes screening.  There are two
5' assay oligos, designed to hybridize upstream of the SNP, with the 3í base of each being
allele-specific. The third oligo is located 3' of the two allele-specific oligos (ASOs), and is a
locus-specific oligo (LSO).  All three oligonucleotide sequences also contain universal PCR
primer sites.  The LSO contains a unique address sequence that is complementary to a capture
sequence on the array. 

LSID:  urn:LSID:illumina.hapmap.org:Protocol:Golden_Gate_assay_design_1.1.0:1
Title: GoldenGateTM Assay Design Protocol
Description: Candidate SNPs first undergo an informatic screen for suitability for assay design.  SNPs
can be excluded from assay design if they are located near or within palindromic sequences,
GT- or AT-rich regions or repeats similar to sequences elsewhere in the genome.
Three assay oligos are designed for each SNP locus that passes screening.  There are two
5' assay oligos, designed to hybridize upstream of the SNP, with the 3í base of each being
allele-specific. The third oligo is located 3' of the two allele-specific oligos (ASOs), and is a
locus-specific oligo (LSO).  All three oligonucleotide sequences also contain universal PCR
primer sites.  The LSO contains a unique address sequence that is complementary to a capture
sequence on the array.

FP-TDI platform (used by UCSF/WashU)
Defined protocols:
LSID:  urn:lsid:ucsf-wu.hapmap.org:Protocol:assay_design:1
Title: Genotyping Assay Design for Single Base Extention and FP-TDI Detection
Description:
 For each SNP two PCR primers and a forward and reverse single base extention SNP
 primer design was attempted. After obtainign uniquely mapped SNPs with other known
 SNPs marked in the sequence the design was attempted in two different steps: 1) SNP
 primer design and 2) PCR primer design.

1) SNP Primer Design

 The TM of the shortest possible (14 bases) was calculated using Nearest Neighbor
 Theromodynamics (Beasley, E.M., R.M. Myers, D.R. Cox and L.C. Lazzeroni. 1999.
 Statistical Refinement of Primer Design Parameters., pp. 55-71. In D.H.G. Michael A.
 Innis, John J. Sninsky ). If the primer TM was less than 50 degrees C the primer was
 extended another base until the TM was between 50-55 degrees (using Allawi and
 SantaLucia Nearest Neighbor Sequence Dependent Thermodynamic Parameters as
 described in Owczarzy, R., Vallone, P.M., Gallo, F.J., Paner, T.M., Lane, M.J. 1997.
 Predicting Sequence-Dependent Melting Stability of Short Duplex DNA Oligomers.
 Biopoly 44: 217-239.) or until the length of the primer as greater than 40 bases.
 If a primer between 14-40 bases long with a TM of 50-55 degrees was found for
 both the forward and reverse SNP primers a preferred SNP primer was chosen to try
 exprimentally by assigning penalties according to the criteria in Table 1. The penalty
 scores are then compared. If the primers have different penalty scores pick the one
 with the lowest. If the penalty scores are the same and the alleles are C/T or G/A pick
 the primer that will result in genotyping C/T (best for FP-TDI), other wise pick the shortest
 primer (cheapest). If a primer is still not picked default to picking forward primer.

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