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 release0.9(Steve Rozen, Helen J. Skaletsky (1996, 1997, 1998) Primer3. Code available athttp://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

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
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 FRETprobes 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.