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.