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PCR Primer Design Following Repeat Masking of sequence, PCR Primer were designed using Primer3 release 0.9 (Steve Rozen, Helen J. Skaletsky (1996,1997,1998) Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html) with similar parameters as described previously (Vieux, E.F., Kwok, P.Y., Miller, R.D.Primer design for PCR and sequencing in high-throughput analysis of SNPs.Biotechniques32:S28-S32), with the minor changes: TARGET= SNP_Positon-20 bases, 20 bases PRIMER_OPT_SIZE=23 PRIMER_MAX_SIZE=26 PRIMER_MIN_SIZE=20 PRIMER_OPT_TM=55 PRIMER_MAX_TM=56 PRIMER_MIN_TM=54 PRIMER_PRODUCT_SIZE_RANGE=80-300 PRIMER_PRODUCT_OPT_SIZE=250 PRIMER_PAIR_WT_PRODUCT_SIZE_LT=.20 PRIMER_PAIR_WT_PRODUCT_SIZE_GT=.20 PRIMER_MIN_GC=20 PRIMER_MAX_GC=50 PRIMER_SALT_CONC=50 PRIMER_SELF_ANY=8 PRIMER_SELF_END=3 PRIMER_DNA_CONC=40 PRIMER_GC_CLAMP=0 PRIMER_MAX_END_STABILITY=8 PRIMER_NUM_RETURN=1
LSID: urn:LSID:imsut-riken.hapmap.org:Protocol:assay_design:1
Title: Design of the Invader Genotyping Assay
Description:
For each SNP two PCR primers and Invader Genotyping Assay was attempted.
After obtaining uniquely mapped SNPs with other known SNPs marked in the sequence,
the design was attempted in two different steps: 1) PCR Primer Design and 2) Invader
Genotyping Assay design.
1) PCR Primer Design
Following Repeat Masking of sequence, PCR Primer were designed using Primer3 release
0.9(Steve Rozen, Helen J. Skaletsky (1996, 1997, 1998) Primer3. Code available at
http://www-genome.wi.mit.edu/genome_software/other/primer3.html) with similar parameters
as described previously (Vieux, E.F., Kwok, P.Y., Miller, R.D. Primer design for PCR and
sequencing in high-throughput analysis of SNPs. Biotechniques 32:S28-S32, with the minor
changes:
TARGET=SNP_Position-23 bases, 47 bases
PRIMER_PRODUCT_SIZE_RANGE=81-600
PRIMER_FIRST_BASE_INDEX=1
PRIMER_GC_CLAMP=1
PRIMER_OPT_SIZE=20
PRIMER_MAX_SIZE=25
PRIMER_MIN_SIZE=17
PRIMER_OPT_TM=60
PRIMER_MAX_TM=64
PRIMER_MIN_TM=58
PRIMER_MAX_DIFF=100
PRIMER_MIN_GC=30
PRIMER_MAX_GC=70
PRIMER_MAX_POLY=3
PRIMER_NUM_RETURN=1000
2) Invader Assay Design
1. To design an Invader assay for SNP genotyping, the sequence of 40-50 bases on
each side of the polymorphic site on the target must be known. Although either the sense
or antisense DNA strand can be used, certain features of the probes, such as four or more
Gs in a row or sequences that might cause the target-specific region of the primary
(signal) probe to form a secondary structure with its 5' flap region, indicate that the
opposite target strand should be used instead.
2. Primary probes used in the Invader assay have a 5' flap and a target-specific
region. The base at the SNP site on the target DNA determines the base at the 5' end of
the target-specific region. In addition, the length of the target-specific region is chosen so
that the Tm of the probe-target duplex is approximately 63 degrees C. The Tm can be calculated
with the Hyther program developed by Peyret and SantaLucia at Wayne State University
(http://jsl1.chem.wayne.edu/Hyther/hythermenu.html) or by any similar program using
nearest-neighbor parameters for DNA (11,12) and including the concentrations of the
probe 1uM. Because the target-specific region of each primary probe will detect only
one polymorphic nucleotide at the SNP site, two unique target-specific regions must be
designed for a typical di-allelic SNP locus. To complete the primary probe design, the
target-specific region is extended at the 5' end with one of the universal 5' flap sequences.
These universal 5' flap sequences are independent from the target sequence. As a result,
practically any SNP assay can use primary probes designed with different target-specific
regions, but the identical two 5' flap sequences. .
3. The design of the invasive probe starts with its 3' terminal nucleotide. That
nucleotide overlaps with the primary probe's target-specific region at the SNP site and
should be non-complementary to the polymorphic nucleotides at the SNP site, following
the order T = C > A > G. Because of this design feature, the identical invasive probe can
be used with both primary probes for a particular target. Except for its 3' terminal
nucleotide, the invasive probe is complementary to the target. The length of the invasive
probe is chosen so that the Tm of the probe-target duplex is approximately of 73-78 degrees C or
10-15 degrees C higher than that of the primary probe.
4.The two FRET cassettes complementing the 5' flaps of the primary probes
complete the design of the Invader assay. Like the 5' flaps, the two FRET cassettes are
designed to be universal; the identical FRET cassettes can be used successfully in
practically any Invader reaction. Both probes use fluorescent dyes FAM and VIC in
combination with a quencher (dabcyl-dT). The 3' ends of the FRET probes are blocked
with an amino group (Glen Research).
Reference:
Single Nucleotide Polymorphisms (Methods and Protocols) Volume 212, Chapter 16,
V. Lyamichev and B. Neri pp.229-240 Humana Press.2002.
LSID: urn:LSID:bcm.hapmap.org:Protocol:assay-design_0001:1 Title: Assay Design for MIP Genotyping Description: For each SNP a single MIP probe is designed. A minimum of 5000 probes are designed as a probe set. 1) MIP probe design Two homologies are designed that abut, but do not overlap the SNP. The tag sequence, two primer sequences and a restriction sequence are added to homology 1 and homology 2 in the order (5' - 3') homology 2, tag sequence, restriction site, primer P2, primer P1, and homology 1.
LSID: urn:lsid:perlegen.hapmap.org:Protocol:LR_design_1.0.0:1 Title: Perlegen Assay Design Protocol Description: Long-range PCR assays were designed using Oligo primer design software (Molecular Biology Insights). Primers were selected to have similar stringency and to map uniquely to NCBI Build 33. From a collection of all suitable candidate primers, we used custom software to select a minimum spanning set having maximum coverage with minimal overlap between adjacent amplicons. Genotyping arrays of 25-bp oligonucleotides were designed as four sets of 20 features (80 features per SNP), corresponding to forward and reverse strand tilings of sequences complementary to each of two SNP alleles. A set of 20 features consisted of five sets of 4 features where the location of the SNP within the oligonucleotide varies from position 11 to position 15. A set of four features consisted of sequences where A, C, T, or G is substituted at position 13. Thus, each set of four features provided one perfect match to the sequence of the corresponding SNP allele and three features with a single-base mismatch for that allele. Mismatch probes were used to measure background, and by comparison with the signal for the perfect match probes, to detect the presence or absence of a specific PCR product.
LSID: urn:lsid:wicgr.hapmap.org:Protocol:assay_design_1:1 Title: Primer and probe design with Sequenom SpectroDESIGNER Description: All genotyping reactions were performed in multiplex format (maximunm 7-fold, minimum 4-fold, average 6.5-fold). For each SNP, a forward PCR primer, a reverse PCR primer and a primer extension probe were designed. SNPs were assorted into multiplex groups and constituent PCR primer and probes were designed using SpectroDESIGNER software version. 2.0 (Sequenom, San Diego, CA). The assay design filter for SNP sequences is 2 alleles. The length of the amplicon (Bp) for SNP capture min 60 opt 90 max 120 with the hME-10 Primary and Secondary tags. Extend primer design length is 17-24 (Bp) with a temperature of 45-100 oC. Peak masses are seperated by a min 42Da with an upper limit at 8250Da.