Fluorescent Genotyping (Simplex PCR, Multiplex Gel)
Purpose: To use a fluorescence based assay to amplify microsatellites quickly, accurately and efficiently. This
assay was developed as a modification of Oetting, W.S., et.al. (1995), to genotype microsatellites using an ABI
377 DNA sequencer.
5’
3’
3’
5’
Double-stranded genomic DNA (gDNA) is denatured and the
M13-tailed forward primer anneals to the complementary strand.
In the following elongation step of PCR, tailed M13 is present in
one strand of the PCR product.
B
5’
3’
3’
5’
M13 tailed forward primer(TGTAAAACGACGGCCAGT)
M13-primer-dye conjugate (TGTAAAACGACGGCCAGT)
In subsequent cycles, the product can be visualized due to the
presence of a binding site for the M13-primer-dye conjugate i.e.
giving the product its end color.
Procedure:
A. PCR Plate Setup
1. Plate 30ng [5ng/ul] of DNA into appropriate wells (one well=one marker=one genotype) according
to the number of markers versus the number of samples to be run (see Example of Plate Layout, pg.
4). To keep the PCR volume low, thereby reducing cost, allow the DNA to dry overnight at room
temperature covered with a breathable tissue (ie. paper towel). Alternately, a speed vac or hot block
can be used.
2. Dilute forward (M13 tailed) and reverse primers, with water, to 460nM, mix together, and dispense
3ul into appropriate wells containing dried DNA. This primer concentration will be 2X the final
reaction concentration.
Plating the mixed primers first allows this portion of the procedure to be done on the bench at room
temperature.
3. Prepare PCR mixes at 2X concentration and pipet, on ice, into appropriate wells (see Example of
Plate Layout, pg. 3).
You will have 2-3 tubes of PCR mix, each with a single M13 fluorophore-conjugated primer, which
contain enough volume to dispense 3ul into all of the wells requiring that fluorophore, (see Pg. 4).
By dispensing the primers into the wells first, you have avoided the need to prepare a separate PCR
mix for each marker, reducing setup time. Final concentration of all reagents in the reaction will be
1X.
K. L. Kennedy
1
4. Carefully seal the plate, centrifuge for 1 minute, and begin thermocycling procedure following the
conditions listed below. To prevent mis-priming, allow the thermocycling heat block to reach a
temperature over 72°C before placing plate on block.
5. When thermocycling is complete, remove the plate, wrap in foil, and store at 4°C until ready to use.
B. PCR Reaction Mix







Genomic DNA at 5ng/ul; 30ng of DNA is used per reaction
230nM forward (M13 tailed) and reverse PCR primer (see below for details on primer design)
PCR Mix
200uM dNTPs (Roche)
1X PCR Buffer, provided with taq (Roche)
150nM M13-dye conjugate (ABI)
5’ Fluorophore-TGTAAAACGACGGCCAGT
Fluorophores used: 6-FAM (B), NED (Y), HEX (G)
0.06-0.24U/ul taq DNA polymerase (Roche)
Note: Titration to determine best taq concentration is highly recommended.
C. Primer design and conditions for use
The forward primer of all marker sets will have an M13 tail attached to the 5’ end. The sequence of the
M13 tail is as follows:
5’ TGTAAAACGACGGCCAGT-XXXXXXXXXXXXXXXX 3’
The forward and reverse primers are mixed equally and are used at a final concentration of 230nM per
marker in the PCR reaction.
K. L. Kennedy
2
D. PCR Thermocycling Conditions: It is recommended that ‘Touchdown PCR’ be used to increase
specificity and reduce background amplification, which is essential to correct allele calling and
critical when pooling markers.
Option 1: Cycling moves from denature to anneal, then back to denature with a gradual step down of the
annealing temperature in between. We have used this method with much success. However, we are
working to shorten the PCR time by making some adjustments shown below in option 2.
Temperature ºC
95
95
68
72
95
58
72
95
54
72
72
4
Time
5 minutes
45 seconds
5 minutes
1 minute
45 seconds
2 minutes
1 minute
45 seconds
2 minutes
1 minute
10 mintues
Hold
Cycles
1X
Cycle 6X; ramp annealing temperature from 68ºC to 60ºC
Cycle 2X; ramp annealing temperature from 58ºC to 56ºC
30X
Option 2: We have condensed the two blocks of the touchdown to one and shortened the annealing time
for each step. This has also worked well for us and has shortened the PCR time by 30-45 minutes.
Temperature ºC
95
95
68
72
95
54
72
72
4
K. L. Kennedy
Time
5 minutes
45 seconds
45 seconds
1 minute
45 seconds
45 seconds
1 minute
10 minutes
Hold
Cycles
1X
7X; ramp annealing temperature from 68ºC to 56ºC
30X
1X
3
Example of Plate Layout: Genomics Component of PGA
The plate design described here is to help new users design their experiments. We use MJ Research 384 well
PCR plates on MJ Tetrad thermocyclers. We are a high-throughput genotyping team, and the assay design
reflects this. However, this assay can be scaled down or adjusted to fit the needs of any lab using any plate or
sequencer. The principles can even be adjusted to single tube assays, but the example to follow is for the larger
experiments.
Note: We use 96-lane ABI-377 DNA sequencers. The gel loader used is a dual position 8-syringe loader
purchased from Kloehn. Basically, one 384- well plate is pooled to condense down to one 96-well plate, which
is then loaded on one gel. The plate design is a product of the need to use this 8-syringe loader for the gels.
After the DNA is extracted, plate 30ng of DNA (6ul @ 5ng/ul) into the appropriate well of a 384-well plate. To
do this, make a deep-well plate containing enough DNA to dispense as many “rows” of a 384-well plate
needed.
Example: Description below
Deep-well
Plate
A
1
5555_1
2
5556_1
3
5557_6
4
5
6
7
8
9
10
11
12
Plate 1 384-well
Chr. 1
5555_1
5555_1
5555_1
5555_1
Chr. 2
5556_1
5557_6
5556_1
5557_6
5556_1
5557_6
5556_1
P24 5557_6
K. L. Kennedy
B
5555_2
5556_2
5557_7
C
5555_3
5556_3
5557_8
D
5555_4
5557_1
BN
E
BN
5557_2
SS
F
G
H
SS
SS HET
Water
5557_3 5557_4 5557_5
SS HET
Water
Water
MJ Res.
Plate
5555_2
5555_2
5555_2
5555_2
5556_2
5557_7
5556_2
5557_7
5556_2
5557_7
5556_2
5557_7
5555_3
5555_3
5555_3
5555_3
5556_3
5557_8
5556_3
5557_8
5556_3
5557_8
5556_3
5557_8
5555_4
5555_4
5555_4
5555_4
5557_1
BN
5557_1
BN
5557_1
BN
5557_1
BN
BN
BN
BN
BN
5557_2
SS
5557_2
SS
5557_2
SS
5557_2
SS
SS
SS
SS
SS
5557_3
SS HET
5557_3
SS HET
5557_3
SS HET
5557_3
SS HET
SS HET
SS HET
SS HET
SS HET
5557_4
Water
5557_4
Water
5557_4
Water
5557_4
Water
Chr1
Chr2
B1
A1
Water 1 thru 4
Water 5 thru 8
Water 9 thru 12
Water 13 and 14
5557_5
1 thru 4
Water
5557_5
5 thru 8
Water
5557_5
9 thru 12
Water
5557_5
13 and 14
Water A24
4
The deep-well plate, illustrated above, is a 96-well plate. Plate 1 is a 384-well PCR plate, shown in proper
orientation. Each square in the representative plate (designed in Excel) is four wells on a 384-well plate (see
image below). Each square contains the same DNA in each of the four wells. Therefore, from the deep well,
each DNA will be plated four times in a quadrant row on the PCR plate. At 6ul/well of DNA x 4 wells=24ul per
well in the deep well x the number of rows (which is dependant on the number of markers to run). In this
example, we are running four animals plus controls for Chromosome 1. Chromosome 1 has 16 markers in the
set. That is then 4 rows x 24ul DNA= 96ul(+extra) for each DNA in your deep well. Chromosome 2 has 12
animals plus controls and 14 markers associated with it. That is also four rows, not complete, in the fourth row
(2 markers run in the last row instead of the normal four). So each DNA will again need to be 96ul + extra.
Plate the DNA and allow drying as described above.
Note: In order to run a Gene Scan gel, a sample sheet must be in place (see software manual for explanation).
The plate design shown in Excel is necessary to use a the macro designed in our lab, from which the sample
sheets for Gene Scan are made. This is a very useful tool and will be posted. The Macro allows for the user to
load in a systematic fashion without having to map out where things end up on the gel. Please refer to the
Sample Sheet Design protocol for more information.
Here is an example of a run-list of markers for this example plate. It shows all the markers by groups of four, as
well as showing the assigned color (In this case, 2-blue, 2-yellow, 2-blue, 2-yellow, etc…). In this scheme, there
are four markers to be pooled together and run in one lane on the gel. Two markers will have the 6-FAM label,
and two markers will have the NED label. Note: Within each color the marker sizes are different from one
another such that one allele set will not interfere with the other. However, different colors can have the same
size, as they can be analyzed separately.
K. L. Kennedy
5
Marker Set for Chr. 1 and 2:
BNxSS
ACP or
D_Name
D1rat167
D1rat4
D1rat234
D1rat154
D1Rat182
D1rat24
D1rat265
D1Rat183
D1rat287
D1rat47
D1rat210
D1rat73
D1mgh13
D1Rat89
BN
229
170
150
238
128
174
159
237
194
158
138
171
139
173
SS
239
160
152
232
140
166
143
227
204
162
152
178
147
202
Abs_Position
0
46.8
274.5
315.6
383
489.2
586.6
666.3
996.5
837.1
732.1
1180
1234.6
1312
F_or_P
F
F
P>3.00
F
F
P>3.00
P>3.00
F
P>3.00
P>3.00
P>3.00
P>3.00
F
P>3.00
D2rat114
D2rat90
D2rat12
D2rat14
D2rat40
D2Mit35
D2rat149
D2rat147
D2rat290
D2rat163
D2rat65
D2mit16
D2rat20
D2mgh11
245
129
156
133
133
266
234
183
244
164
164
188
180
220
239
123
159
141
129
258
253
192
250
148
146
194
172
214
0
150.2
54.2
239.7
777
633
847.7
695.5
1086.1
456.6
1156.6
1201.4
340.6
965.5
F
F
P>3.00
F
F
F
P>3.00
P>3.00
P>3.00
F
F
P>3.00
P>3.00
F
1 thru 4
5 thru 8
9 thru 12
13 and 14
1 thru 4
5 thru 8
9 thru 12
13 and 14
We always begin PCR set-up by plating the primers first. Dispense 3ul of 0.46uM primer into appropriate wells
containing dried DNA. This can be done right on the bench. A black surface is helpful in seeing where the
primer is in the plate.
First row of a 384-well plate:
p
o
n
m
c
k
j
i
h
g
A1 (Top Right
f
e
d
c
b
a
1
2
1
2
1
2
1
2
1 2
1
2
1
2
1
2 Blue PCR mix
3
4
3
4
3
4
3
4
3 4
3
4
3
4
3
4 Yellow PCR mix
Each quadrant of four is represented on plate 1 by one block. There is actually 16 wells in one
row. Well #1 is one marker. Well #2 is another marker, so on…
K. L. Kennedy
6
Once you have the primers dispensed, place the plate on ice. The way we have the plates designed for pooling
multiple markers is as follows: In a grid of four wells (i.e. A1, A2, B1, and B2) The top two wells (A1, A2) are
Blue, using the 6-FAM M13 dye conjugate in the PCR mix. The bottom two wells (B1, B2) are Yellow or
Green, using the NED or HEX M13-dye conjugate, respectively. In this scheme, two colors per plate would
alternate every other row (blue, green, blue, green etc…). Plate all the PCR mix for one color and then follow
with the other color until the whole plate has PCR mix in the appropriate wells.
The plate is now ready for PCR. Follow the directions above for preparing the plate for PCR and thermocyler
conditions.
Summary of Set-up:






Prepare DNA at 5ng/ul
Design plates in Excel
Plate DNA into 384 well plates from a 96-well deep well, allow to dry
Add primers to appropriate wells
Add PCR mix
Spin, seal, and PCR
What comes next?

Purification
K. L. Kennedy
7