Genotyping of short tandem repeats (STRs) markers with 6 bp or higher length difference using PCR and high resolution agarose electrophoresis
1
Laboratory of Molecular and Cellular Immunology, Institute of Molecular Genetics AS CR, v.v.i.,
Prague, Czech Republic. 2Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech Republic. Correspondence should be addressed to M.L. (
[email protected]).
This protocol describes DNA typing of short tandem repeats (STRs), which differ in at least 6 bp using PCR and optimized high resolution electrophoresis. DNA preparation by NaOH method or by standard TRI reagent procedure takes about 4 hours. When DNA is prepared in master plates, a single person can test 192 samples in maximally 5 hours. It is suitable for quick test of interval-specific congenic strains, marker-assisted breeding of congenic mouse strains, test of presence of transgenes, knock-out or knock-in alleles in gene targeting technologies in breeding crosses, and for genotyping of intraspecific crosses, especially those derived from parents, which differ in limited percentage of their genomes.
KEYWORDS
2 INTRODUCTION
3
4
5
MATERIALS
REAGENTS
●
! CAUTION
●
! CAUTION
●
●
! CAUTION
6 ●
●
●
! CAUTION
! CAUTION
! CAUTION
● ●
●
! CAUTION
7
●
! CAUTION
●
●
! CAUTION
! CAUTION
● ●
●
! CAUTION
! CAUTION
8 ●
! CAUTION
● ●
●
! CAUTION
● ● ● ●
! CAUTION
●
●
! CAUTION
9 ●
! CAUTION
●
! CAUTION
●
! CAUTION
●
●
●
! CAUTION
! CAUTION
! CAUTION
10 ●
● ● ● ●
● ● ●
! CAUTION
11 ● ● ● ●
● ● ● ●
● ● ●
● ●
12
13
14
▲ CRITICAL STEP
▲ CRITICAL STEP
15
16
! CAUTION
1|
17
● TIMING
● TIMING
■ PAUSE POINT
● TIMING
▲ CRITICAL STEP
18
■ PAUSE POINT
19
● TIMING
■ PAUSE POINT
2|
▲ CRITICAL STEP
20
● TIMING
▲ CRITICAL STEP
● TIMING
● TIMING
21 ▲ CRITICAL STEP
■ PAUSE POINT
3|
● TIMING ▲CRITICAL STEP
▲CRITICAL STEP
■ PAUSE POINT
22
● TIMING
▲ CRITICAL STEP
▲ CRITICAL STEP
? TROUBLESHOOTING
23 ANTICIPATED RESULTS
24 ACKNOWLEDGMENTS
COMPETING FINANCIAL INTERESTS
25 REFERENCES
26
27 TABLES
number of reactions* Forward primer 6.7 μM Reverse primer 6.7 μM 5x PCR buffer total Sterile distilled H2O Taq polymerase 1U/μl Total volume
1 6 (9) 9 (13) 12 (15) 14 (18) 18 (27) 24 (30) 24 (36) 37 (45) 36 (54) 48 (60) 0.34 3.06 4.4 5.1 6.1 9.2 10.2 12.2 15.3 18.4 20.4 0.34 3.06 4.4 5.1 6.1 9.2 10.2 12.2 15.3 18.4 20.4 4 36 52 60 72 108 120 144 180 216 240 4.9 44.3 64.0 73.8 88.6 132.8 147.6 177.1 221.4 265.7 295.2 0.4 3.6 5.2 6.0 7.2 10.8 12.0 14.4 18.0 21.6 24.0 10 90 130 150 180 270 300 360 450 540 600 units: μl
* number in brackets = theoretical number of reactions (without loss due to sticking of the liquid on the surface of tubes and pipettes)
28
1
2
3
4
5
6
7
8
9
10
11
12
A
spl1
3
5
7
9
11
13
15
17
19
21
23
B
2
4
6
8
10
12
14
16
18
20
22
24
C
25
27
29
31
33
35
37
39
41
43
45
47
D
26
28
30
32
34
36
38
40
42
44
46
48
E
49
51
53
55
57
59
61
63
65
67
69
71
F
50
52
54
56
58
60
62
64
66
68
70
72
G
73
75
77
79
81
83
85
87
89
91
93
95
H
74
76
78
80
82
84
86
88
90
92
94
96
29
Problem There is too much product at start of electrophoresis
There is no product
Possible reason Too much DNA in sample
Solution Dilute DNA sample before repeating of PCR
Bad conditions of reaction for certain primers
Optimize reaction conditions, usually different annealing temperature or Mg2+ concentration is necessary Use DNA from stock solution or isolate new DNA Optimize reaction conditions, usually usually different annealing temperature or different Mg2+ concentration is necessary Use new chemicals Prevent contamination (use gloves, work in biohazard box etc.) Optimize reaction conditions, usually different annealing temperature or Mg2+ concentration is needed
Bad quality of DNA sample Bad conditions of reaction for certain primers Bad quality of chemicals Contamination of solutions
There are non-specific products overlap with PCR product
Bad conditions of reaction for certain primers.
30 FIGURES
