SNP SCALE Protocol.
Overview
SNP SCALE (SNP Scoring by Colour And Length Exclusion) is a SNP genotyping
system based on allele specific PCR. Allele specific PCR relies on differences in the
binding efficiencies of a perfect match versus a mismatch for each of the allele specific
forward primers to distinguish between the two SNP alleles. The advantages of allele
specific PCR include all of the normal benefits associated with PCR: high sensitivity,
repeatability, can work on small amounts of tissue and degraded tissue, rapid, and
flexible. However there are some problems unique to allele specific PCR for SNP
genotyping, in particular the cost of fluorescent primers and potential errors associated
with non-specific primer binding. SNP SCALE has been designed to take advantage of
the benefits of allele specific PCR, while trying to overcome some of the drawbacks.
SNP SCALE uses a specially modified oligonucleotide, Locked Nucleic Acids (LNAs),
in the 3’ SNP position to overcome the problems of non-specific binding, and it uses 5’
tailing and universal fluorescent oligos (UFOs) to overcome the problems of fluorescent
labelling costs. The 5’ tails, which are slightly different in length, have the added
advantage of facilitating allele scoring by both colour and length exclusion.
Described here is the SNP SCALE protocol, including PCR set-up and reaction
conditions. Also discussed are issues associated with primer design. Because there are
five primers in the reaction mixture, there are 25 possible forward and reverse primer
combinations, exponentially increasing the chance of non-specific binding. Thus good
primer design is critical.
LNAs (Locked Nucleic Acids)
LNAs are a specially modified nucleic acid that increase primer binding specificity, and
thus decrease mis-priming. Also they raise the effective annealing temperature of the
primer by about three degrees, so that the effective difference in annealing temperature
between the allele specific primers is further increased. For example a primer that
typically anneals at 50 degrees will anneal at 53 – 55 degrees.
UFOs (Universal Fluorescent Oligos)
One drawback of allele specific PCR is the cost of two fluorescent primers per SNP
locus, one per allele. Furthermore SNPs are less polymorphic than microsatellites and
thus less informative. Therefore more markers are required for the same information
content, further increasing costs. We have largely overcome this problem by using a
universal fluorescent labelling system. Each allele specific LNA oligo is tailed with one
of two different M13 sequences identical in sequence to its corresponding universal
fluorescent oligo (UFOs). The UFOs can then be incorporated during PCR, as explained
below.
The SNP SCALE process
SNP SCALE is a homogenous two step process: allele specific amplification, and allele
specific labelling (Fig 1). The PCR setup contains 5 primers: two 5’ tailed allele specific
LNA oligos (LNAs), two universal fluorescent oligos (UFOs), and a single common
reverse oligo (CRO). In phase one, the LNA-ASOs bind with high specificity to their
corresponding alleles, while the CRO binds to the reverse template of both alleles and
allele specific amplification begins. At this stage the temperature is too high for the UFOs
to bind, preventing non-specific binding. During PCR, the CRO incorporates the 5’ tails
into the template as it amplifies templates previously generated by the tailed allele
specific LNAs, thus generating priming sites for the UFOs.
After 10-15 cycles, the annealing temperature is lowered sufficiently such that the UFOs
can bind and phase two, allele specific labelling, commence. Despite the lower
temperature, mis-priming remains minimal due to the high template to background ratio
and because the amount of each LNA added to the PCR is limited (1/10th concentration)
so that it will run out during phase one and be unavailable for amplification at the lower
temperature.
Fig. 1: SNP-SCALE process overview.
Protocol
Table 1: SNP SCALE setup for a 10 ul PCR
Reagent
PCR Buffer
dNTPs
LNA-ASO 1
LNA-ASO 2
UFO 1 (FAM)
UFO 2 (HEX)
CRO
MgCl2
Taq
Water
[stock]
10 x
2 mM
0.2 u M
0.2 u M
1 uM
1 uM
2 uM
50 mM
5 U/u l
NA
[PCR]
1x
0.2 mM
0.02 u M
0.02 u M
0.1 u M
0.1 u M
0.2 u M
2 mM
0.5U
NA
DNA
0.1 u g/u l
0.01 u g/u l
Reaction Volume
1
1
1
1
1
1
1
0.4
0.05
1.6
1
Table 2: SNP SCALE PCR reaction:
Phase
1
2
Temperature
94
Time
03:00
Cycles
94
60-1/cycle
72
00:30
00:30
01:00
10
94
50
72
00:30
00:30
01:00
30
72
10
05:00
01:00
END
Primer Design
Good primers are critical for good genotypes in SNP SCALE. So I will discuss here in
some detail primer design.
We use Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to design
primers. There are many parameters to consider in primer design, listed below.
PCR primers are designed with the following criteria
Table 3: SNP-SCALE primer design criteria.
Parameter
PCR Product
Primer Tm
Self Complimentarity
GC clamp
Pigtail
Mismatches (primer SNPs)
Maximum 3' Stability
Primer Size
Min
Max
Opt
Total
3'
Min
Max
Opt
LNA
CRO
80-550
80-550
58
63
62
67
60
65
8
8
3
3
no
yes
no
yes
0
0
9
9
18
18
25
25
20
20
The two most critical parameters are Primer Tm and Self Complimentarity. The LNA
allele specific oligos (LNA-ASOs) should be designed with an annealing temperature as
close to 60 degrees as possible, while the CROs should be designed with an annealing
temperature as close to 65 degrees as possible. The difference in annealing temperatures
is due to the inclusion of a 3’ LNA base, which effectively raises the annealing
temperature of the LNA-ASO to that of the CRO. It is also important to remember that
the position of the LNA-ASO is restricted; it MUST end on the SNP site, and there
shouldn’t be any other SNPs in the rest of the primer (although of course the primer can
be in either forward or reverse orientation on either side of the SNP). Note that a Tm of
65 on Primer 3 corresponds in practice to a PCR annealing temperature (Ta) of about 55
degrees. Although the optimal annealing temperature will change slightly for each pair of
LNAs, it should remain constant for the UFOs.
One problem often encountered in PCR, particularly with microsatellites, is nontemplated addition of a single adenine base at the 3’ end of the sequence (+A addition).
This phenomenon can also occur in SNP-SCALE (see figure 2) and may interfere with
accurate allele scoring. To avoid this we recommend appending a “pigtail” sequence to
the 5’ end of the reverse primer. This will ensure that +A addition is standard across loci
and across alleles. The pigtail primer sequence is: GTTTCTT.
Once you have designed a forward and reverse primer you must then decide which tail to
place on which primer. Currently we have a choice of only two tails, but there is no
reason you can’t expand this set, giving you more options for better primer design while
still allowing colour and length exclusion. The two tails are:
Tail1_UFO_HEX: CAGGGTTTTCCCAGTCACGAC (21bp)
Tail2_UFO_FAM: AGCGGATAACAATTTCACACAGGA (24bp)
A new set of UFOs have now been developed and tested for their relative migration rates
with different colour labels (Table 4).
Table 4. Universal fluorescent primers (UFOs). Relative migration is relative observed
size of PCR products with each UFO compared to UFO3-FAM.
Name with
dye
UFO1-HEX
UFO1-VIC
UFO2-FAM
UFO3-FAM
UFO3-PET
UFO4-FAM
UFO4-NED
UFO5-HEX
UFO5-NED
UFO6-HEX
UFO7-HEX
UFO7-NED
UFO7-PET
5' - 3'
mer
Relative migration (bp)
CAGGGTTTTCCCAGTCACGAC
Same
AGCGGATAACAATTTCACACAGGA
AGCCGTTGCTACCCTCGTTC
Same
GTTCTGAGGGTGGCGGTTCT
Same
CATGGGTTCCTATTGGGCTTG
Same
GCAAAACCCCGCTAATCCTAATC
AATCAGTGAGGCCACCGAGTAAA
Same
Same
21
Same
24
20
Same
20
Same
21
Same
23
23
Same
Same
0.7 - 1.5
1.1 - 2.4
3.5 - 4.2
0
2.4 - 4.0
0.2 - 0.6
0.4 - 0.9
1.7 - 2.1
1.6 - 2.1
2.6 - 3.3
2.7 - 3.3
2.9 - 3.9
5.4 - 6.3
SNP Genotyping
After the PCR reaction, PCR products are separated on the ABI3730, following standard
protocols for genotyping microsatellites. A typical SNP product for a single locus will
look like this:
Fig. 2: SNP-SCALE genotypes for three individuals at a single SNP locus. The top
genotype is homozygous for the HEX (green) labelled allele. The middle genotype is
heterozygous for both alleles, while the bottom genotype is homozygous for the FAM
(Blue) labelled allele. The double peak for each colour is due to plus-A addition,
commonly seen in microsatellites. This can be overcome by using a pig-tail on the CRO.
Since the SNPs take up a small and known size space on a gel, it is possible to screen
multiple SNPS in one run, saving time and money. In theory you should be able to space
SNPs at intervals of only 7-10bp. Thus it may be possible to fit up to 50 SNPs in a single
gel. When screening multiple SNPs it may be better to use a multiplex protocol and a
four dye system; two different dye pairs per adjacent SNP. Kenta has led the efforts to
develop this advancement and has now submitted a paper for publication. An example of
a cluster of 21 SNPs is shown below:
Fig. 3: SNP genotype chromatogram for multiplex PCR of 21 SNP loci.
SNP-Scoring
Once all samples have been amplified and run on the 3730, it is necessary to score
genotypes. While it is possible to score each manually, a faster and perhaps more
accurate method is to score all genotypes using clustering methods. We have developed a
clustering macro that can be used in excel to semi-automatically score SNP genotypes.
The macro is currently hosted on Jon Slates personal web page (www.jonslate.staff.shef.ac.uk/_Publications/publications.shtml, Hinten et al. 2007).
The macro will produce three types of cluster plot: Cartesian, polar, and peak intensity.
An example of peak intensity plots, is shown below.
Fig. 4: Peak intensity plots for two SNP loci. SNP A shows tight clustering, while SNP B
shows broad clustering, although clusters are still clearly defined.
For polar plots, the heterozygous peak should all cluster around /4 (0.785). For the peak
intensity plots, the heterozygotes should cluster around 0.5.
Cluster quality (validity) can be quantified using silhouette scores, using the ClusterA
program, freely available at: http://www.medsci.uu.se/molmed/software.htm