Background
Materials and Methods
Blood samples
DNA template preparation
Amplification protocols
Gene | Primer | Sequencea | mM Mg2+ b | Annealing °C b | Cyclesb | |||
---|---|---|---|---|---|---|---|---|
1st | 2nd | 1st | 2nd | 1st | 2nd | |||
Pvcs
| VCS-OF | ATGTAGATCTGTCCAAGGCCATAAA | 1 | 58 | 25 | |||
VCS-OR | TAATTGAATAATGCTAGGACTAACAATATG | 1 | 58 | 25 | ||||
VCS-NF | GCAGAACCAAAAAATCCACGTGAAAATAAG | 1 | 62 | 30 | ||||
VCS-NR | CCAACGGTAGCTCTAACTTTATCTAGGTAT | 1 | 62 | 30 | ||||
Pvmsp1
F1
| VM1-O1R | CCACTCCATGAAACTGAAGTGTTTA | 2 | 68 | 25 | |||
VM1-N1F | CGATATTGGAAAATTGGAGACCTTCATCAC | 2 | 2 | 68 | 68 | 25 | 30 | |
VM1-N1R | CTTTTGCGCCTCCTCCAGCTGGCTCGTGT | 2 | 68 | 30 | ||||
Pvmsp1
F2
| VM1-O2F | GATGGAAAGCAACCGAAGAAGGGAAT | 1 | 50 | 25 | |||
VM1-O2R | AGCTTGTACTTTCCATAGTGGTCCAG | 1 | 1 | 50 | 64 | 25 | 35 | |
VM1-N2F | AAAATCGAGAGCATGATCGCCACTGAGAAG | 1 | 64 | 35 | ||||
Pvmsp1
F3
| VM1-3F | CAAGCCTACCAAGAATTGATCCCCAA | 1 | 66 | 42 | |||
VM1-3R | ATTACTTTGTCGTAGTCCTCGGCGTAGTCC | 1 | 66 | 42 |
Analysis of the amplification product
Results
Specificity and sensitivity of the amplification reactions
Genotyping using Pvcs
Allele | Sizea | Pre-repeatb | Post-repeatb | n | Frequencyc |
---|---|---|---|---|---|
VK210a
| A | Yes | No | 1 |
0.008
|
VK210b
| B | Yes | No | 8 |
0.067
|
VK210c
| C | Yes | No | 7 |
0.058
|
VK210d
| C | No | No | 1 |
0.008
|
VK210e
| C | Yes | Yes | 1 |
0.008
|
VK210f
| D | Yes | No | 24 |
0.200
|
VK210g
| D | No | No | 10 |
0.083
|
VK210h
| D | No | Yes | 4 |
0.033
|
VK210i
| E | Yes | No | 22 |
0.183
|
VK210j
| E | No | No | 7 |
0.058
|
VK210k
| E | No | Yes | 3 |
0.025
|
VK210l
| F | No | No | 2 |
0.017
|
VK210m
| F | No | Yes | 1 |
0.008
|
VK210n
| F | Yes | No | 2 |
0.017
|
VK210o
| B | No | No | 2 |
0.017
|
VK210p
| B | No | Yes | 2 |
0.017
|
VK210q
| G | No | No | 1 |
0.008
|
VK210r
| C | No | Yes | 1 |
0.008
|
VK247a
| B | In | ND | 3 |
0.025
|
VK247b
| C | In | ND | 2 |
0.017
|
VK247c
| D | In | ND | 4 |
0.033
|
VK247d
| B | No | ND | 1 |
0.008
|
VK247e
| E | No | ND | 1 |
0.008
|
Genotyping using Pvmsp1
F1 | F2 | F3 | ||||||
---|---|---|---|---|---|---|---|---|
Variant | n | Frequency | Varianta | n | Frequency | Variant | n | Frequency |
A
| 4 |
0.038
|
Aa1
| 50 |
0.5
|
A
| 1 |
0.009
|
B
| 19 |
0.183
|
Aa3
| 6 |
0.06
|
B
| 12 |
0.112
|
C
| 44 |
0.423
|
Aa6
| 15 |
0.15
|
C
| 12 |
0.112
|
D
| 22 |
0.212
|
Aa7
| 4 |
0.04
|
D
| 82 |
0.766
|
E
| 15 |
0.144
|
Aa8
| 1 |
0.01
| |||
Aa9
| 1 |
0.01
| ||||||
Aa10
| 1 |
0.01
| ||||||
Ba1
| 6 |
0.06
| ||||||
Ba2
| 1 |
0.01
| ||||||
Ba3
| 5 |
0.05
| ||||||
Ba5
| 2 |
0.02
| ||||||
Ba6
| 4 |
0.04
| ||||||
Ba7
| 3 |
0.03
| ||||||
Ba10
| 1 |
0.01
| ||||||
Am1
| 38 |
0.38
| ||||||
Am2
| 6 |
0.06
| ||||||
Am3
| 2 |
0.02
| ||||||
Am4
| 4 |
0.04
| ||||||
Am5
| 11 |
0.11
| ||||||
Am6
| 2 |
0.02
| ||||||
Am8
| 14 |
0.14
| ||||||
Am9
| 1 |
0.01
| ||||||
Bm1
| 6 |
0.06
| ||||||
Bm2
| 3 |
0.03
| ||||||
Bm3
| 7 |
0.07
| ||||||
Bm4
| 2 |
0.02
| ||||||
Bm5
| 3 |
0.03
| ||||||
Bm8
| 1 |
0.01
|
Variant | Size | Alu I Pattern number | Mnl Pattern number | na | Frequency |
---|---|---|---|---|---|
Aa
| A | 1 | 1 | 27 |
0.27
|
Ab
| A | 1 | 2 | 5 |
0.05
|
Ac
| A | 1 | 4 | 3 |
0.03
|
Ad
| A | 1 | 5 | 1 |
0.01
|
Ae
| A | 1 | 6 | 1 |
0.01
|
Af
| A | 1 | 8 | 12 |
0.12
|
Ag
| A | 1 | 9 | 1 |
0.01
|
Ah
| A | 3 | 1 | 4 |
0.04
|
Ai
| A | 3 | 2 | 1 |
0.01
|
Aj
| A | 3 | 4 | 1 |
0.01
|
Ak
| A | 6 | 1 | 3 |
0.03
|
Al
| A | 6 | 3 | 2 |
0.02
|
Am
| A | 6 | 5 | 8 |
0.08
|
An
| A | 6 | 6 | 1 |
0.01
|
Ao
| A | 6 | 8 | 1 |
0.01
|
Ap
| A | 7 | 1 | 2 |
0.02
|
Aq
| A | 7 | 5 | 2 |
0.02
|
Ar
| A | 8 | 1 | 1 |
0.01
|
As
| A | 9 | 1 | 1 |
0.01
|
At
| A | 10 | 8 | 1 |
0.01
|
Ba
| B | 1 | 1 | 2 |
0.02
|
Bb
| B | 1 | 2 | 1 |
0.01
|
Bc
| B | 1 | 3 | 1 |
0.01
|
Bd
| B | 1 | 4 | 1 |
0.01
|
Be
| B | 1 | 8 | 1 |
0.01
|
Bf
| B | 2 | 1 | 1 |
0.01
|
Bg
| B | 3 | 1 | 2 |
0.02
|
Bh
| B | 3 | 2 | 1 |
0.01
|
Bi
| B | 3 | 3 | 1 |
0.01
|
Bj
| B | 3 | 4 | 1 |
0.01
|
Bk
| B | 5 | 1 | 1 |
0.01
|
Bl
| B | 5 | 2 | 1 |
0.01
|
Bm
| B | 6 | 3 | 4 |
0.04
|
Bn
| B | 7 | 3 | 1 |
0.01
|
Bo
| B | 7 | 5 | 2 |
0.02
|
Bp
| B | 10 | 5 | 1 |
0.01
|