Detection of known thalassemia point mutations by snapback single-strand conformation polymorphism: The feasibility analysis

Clinical Biochemistry 39 (2006) 833 – 842

Detection of known thalassemia point mutations by snapback single-strand conformation polymorphism: The feasibility analysis☆ Wei Li a,⁎, Feng Gao b , Weizhong Tang b , Xuerong Zhang a , Haitian Zhang c a

Medical Science Research Center, Guangxi Medical University, No. 22, Shuangyong Road, Nanning 530021, Guangxi, PR China b Department of Anal and Colorectal Surgery, The First Affiliated Hospital of Guangxi Medical University, No. 22, Shuangyong Road, Nanning 530021 Guangxi, PR China c Department of Gastrointestinal and Glandular Surgery, The First Affiliated Hospital of Guangxi Medical University, No. 22, Shuangyong Road, Nanning 530021 Guangxi, PR China Received 1 January 2006; received in revised form 6 April 2006; accepted 19 May 2006 Available online 26 May 2006

Abstract Objectives: To explore if snapback single-strand conformation polymorphism (snapback SSCP) can be applied to the detection of known thalassemia point mutations. Design and methods: We examined 13 types of known thalassemia point mutations by using snapback SSCP. Results: These 13 different thalassemia point mutations could be identified by snapback SSCP. Conclusions: Snapback SSCP can be applied to the detection of known thalassemia point mutations. The advantages of snapback SSCP are (1) it is simple as compared to PCR-ASO; (2) snapback SSCP is specific and stable once the conditions of snapback SSCP are optimized; (3) samples can be checked at any time without the limit of half-life of radioactive isotope; and (4) since PCR products used in snapback SSCP can be checked by using 2% agarose gel before snapback SSCP, misdiagnoses caused by false positive or false negative PCR can be reduced significantly in snapback SSCP. © 2006 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: SSCP; β-Thalassemia; α-Thalassemia; Point mutation; Haemoglobinopathy; Monogenic disease; PCR; PAGE; Silver stain; Prenatal diagnosis

Introduction Point mutations in some genes can cause monogenic diseases such as thalassemia, hemophilia, and glucose-6phosphate dehydrogenase deficiency, etc. Several methods for the detection of point mutations have been developed. Known point mutations can be detected by polymerase chain reaction-allele-specific oligonucleotide (PCR-ASO) [1], reverse dot blot hybridization (RDB) [2], the amplification refractory mutation system (ARMS) [3], ligase chain reaction (LCR) [4], restriction enzyme (RE)-PCR [5] or gene chip [6],

☆ This research was supported by Guangxi Natural Specific Fund (No: GKJ 0575063). ⁎ Corresponding author. E-mail address: [email protected] (W. Li).

etc. Unknown point mutations can be screened by using gradient gel electrophoresis (DGGE) [7], denaturing highperformance liquid chromatography (DHPLC) [8], singlestrand conformation polymorphism (SSCP) [9], or DNA sequencing, etc. Each method has its own advantages and disadvantages according to the “Best Practice Guidelines for carrier identification and prenatal diagnosis of Haemoglobinopathy (2002)” of the European Molecular Genetics Quality Network (EMQN, website: http://www1.emqn.org/index.html). For example, PCR-ASO is one of the most common methods used in prenatal diagnosis for monogenic disease owing to its high specificity and reliability, but it is time-consuming and needs radioactive isotopes. ARMS is a simple method, but its specificity is not as high as that of PCR-ASO. It is usually used together with other methods to reduce misdiagnosis caused by false positive or false negative PCR. SSCP is a cost-effective method to screen unknown point mutations. However, some

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W. Li et al. / Clinical Biochemistry 39 (2006) 833–842

point mutations could not be detected by SSCP. Wilton SD and colleagues have developed the snapback SSCP method to detect known mutations which could not be identified with the use of SSCP [10]. When we screened point mutations in several different genes by using SSCP, we found that two complementary sequences in a DNA single-strand could hybridized with each other [11], which seems to support Wilton's findings. An ideal method used in the diagnosis of monogenetic disease should not only be simple but also reliable. Since snapback SSCP is simple and specific according to reference 10, we think it is interesting and useful to explore if snapback SSCP can be applied for the detection of known thalassemia point mutations. Methods Samples 11 Types of known β-thalassemia point mutations and 2 types of known α-thalassemia mutations were used to study the feasibility of snapback SSCP. These mutations had previously been determined by using the PCR-ASO method or DNA sequencing. 11 types of known β-thalassemia point mutations checked were − 28 (A→G), − 29 (A→G), codon 17 (A→T), codon 26 (G→A, HbE), codon 30 (A→G), codons 41–42 (–TTCT), codon 43 (G→T), codons 71–72 (+ A), codon 95 (+ A), IVS-I-1 (G→T) and IVS-II-654 (C→T). Among them, − 28 (A→G), − 29 (A→G), codon 17 (A→T), codon 26 (G→A, HbE), codon 30 (A→G), codon 43 (G→T), IVS-I-1 (G→T) and IVS-II-654(C→T) were base substitution mutations. Codons 41–42 (–TTCT) was a 4-bp deletion mutation. Codons 71–72 (+ A) and codon 95 (+ A) were 1-bp insertion mutations. Codon 17 (A→T) and codons 41–42 (–TTCT) included both heterozygote and homozygote. The other β-thalassemia mutations were heterozygous. 2 types of known α-thalassemia point mutations checked were Hb Constant Spring (terminating codon (TAA→CAA)) and Hb Quong Sze (codon 125 (CTG→CCG)). Both of them were base substitution mutations. Hb Constant Spring included heterozygote and double heterozygote for Hb Constant Spring and --sea. Hb Quong Sze included heterozygote and double heterozygote for Hb Quong Sze and --sea. The mutation, --sea, was a large deletion of the whole α-globin gene cluster. DNA was extracted by using standard the phenol/chloroform method. Nested PCR We used nested PCR to improve the background and sensitivity of snapback SSCP. A total of 59 PCR fragments were amplified. Amongst them, two fragments were the first round PCR products used as templates for the second round PCR. The other 57 fragments were the second round PCR products (68–332 bp long). The sequences of PCR primers are listed in Table 1. To detect point mutations, 7- to 16base long oligonucleotides which were complementary to the mutative or the normal sequences were linked to the 5′-ends

of some internal primers. We call these oligonucleotides intramolecular hybridization (IMH) probes because the snapback of complementary sequence is actually a process of IMH between two complementary sequences in a single DNA strand. Every point mutation was set in the middle of a probe. IMH probes could be linked to 5′-end of either sense strand primers or antisense strand primers, called sense strand probes or antisense strand probes, respectively. Every point mutation was checked by using two or more than two IMH probes until it was identified. A primer linked by an IMH probe is called snapback PCR primer. PCR volume was 10 μL containing 50 mM KCl, 10 mM Tris–HCl (pH 8.8 at 25°C), 0.08% Nonidet P40, 1.5 mM MgCl2, 0.125 mM each dNTP, 10 pmol each primer, 0.4 U of Taq DNA polymerase (MBI Fermentas, USA) and templates (0.2 μg of genomic DNA for the first round PCR, or 1 μL of the first round PCR products diluted 1500fold for the second round PCR). PCR was performed in a PCR cycler (Gene cyclerTM, Bio-Rad, USA). Fragments βFD and α-89 listed in Table 1 were the first round PCR products. The cycling conditions were (1) 95°C for 5 min followed by 35 cycles at 94°C, 30 s, 55°C, 30 s, 72°C, 4 min for the amplification of β-FD; (2) 95°C for 5 min followed by 35 cycles of 94°C, 1 min, 65°C, 1.5 min for the amplification of α-89; (3) 95°C for 3 min followed by 35 cycles of 94°C, 30 s, 53°C, 30 s and 72°C, 1.5 min for the second round PCR. The second round PCR products were checked by using 2% agarose gel with 0.5 μg/mL ethidium bromide and quantitated roughly with a DNA marker, GeneRuler™ 50 bp DNA ladder (MBI Fermentas, USA). The second round PCR products used in snapback SSCP were called snapback SSCP fragments. Principle of snapback SSCP Once a DNA single-strand containing an IMH probe is generated, IMH between probe and its complementary sequence will occur spontaneously. IMH can be interfered by a mismatching base pair between probe and its complementary sequence. When electrophoretic temperature is raised to some degree, IMH with mismatching will be separated, but IMH without mismatching cannot be separated at the same temperature. In discontinuous pH PAGE, the migration rate of DNA single strand with IMH is different from that of DNA single strand without IMH owing to their different conformations. Therefore, a mutation occurring in the middle of the sequence covered by an IMH probe can be identified according to its specific DNA band pattern in discontinuous pH PAGE (Fig. 1). Electrophoresis The concentrations of mini-polyacrylamide gel (mini PAG, 10 × 10 cm2) were 12–20% containing 10% glycerol. The ratio of acrylamide to bisacrylamide was 35:1. The gel was 0.3 mm thick. Tank buffer used was 0.025 M Tris–0.088 M L-glycine, pH8.8. Gel buffer used was 0.112 M Tris–0.112 M

Table 1 PCR fragments, PCR primers, IMH probes, temperature, and the results of snapback SSCP Primers a

PCR fragments No β-FD α-89e

Length (bp) e

Mutations checked

1947 326 96

−28 (A–G)

2

154

−28 (A–G)

3

94

−28 (A–G)

4

93

−28 (A–G)

5

151

−28 (A–G)

6

151

−28 (A–G)

7

148

−28 (A–G)

8

96

−29 (A–G)

9

151

−29 (A–G)

10

93

−29 (A–G)

11

148

−29 (A–G)

12

118

CD17 (A–T)

13

113

CD17 (A–T)

14

153

CD17 (A–T)

15

332

CD17 (A–T)

16

150

CD26 (G–A)

17

90

CD26 (G–A)

18

139

CD30 (A–G)

19

136

CD30 (A–G)

20

82

CD30 (A–G)

21

141

IVS-I-1 (G–T)

Name

Sequences (5′→3′)

βF βD α8 α9 − 28M β1 − 28M β7 − 28M1 β1 − 28M2 β1 − 28M2 β7 − 28N β7 − 28N1 β7 − 29M β1 − 29M β7 − 29M1 β1 − 29M1 β7 17M β50 17M1 β50 17M2 17M1B 17N β50 26M 26R 26M1 β50 30M 26R 30M1 26R 30M β50 1M

AGTAGCAATTTGTACTGATGGTATGG TTTCCCAAGGTTTGAACTAGCTCTT GCTGCGGGCCTGGGCCGCAC CATTGTTGGCACATTCCGGGAC gggcatagaagtcaTAAGCAATAGATGGCTCTg GTTGGCCAATCTACTCCC gggcatagaagtcaTAAGCAATAGATGGCTCTg CCAAGGACAGGTACGGCTGTCATC ggcatagaagtcTAAGCAATAGATGGCTCTg GTTGGCCAATCTACTCCC gcatagaagtcTAAGCAATAGATGGCTCTG GTTGGCCAATCTACTCCC gcatagaagtcTAAGCAATAGATGGCTCTG CCAAGGACAGGTACGGCTGTCATC gggcataaaagtcaTAAGCAATAGATGGCTCTG CCAAGGACAGGTACGGCTGTCATC ggcataaaagtcTAAGCAATAGATGGCTCTG CCAAGGACAGGTACGGCTGTCATC gggcatgaaagtcaTAAGCAATAGATGGCTCTG GTTGGCCAATCTACTCCC gggcatgaaagtcaTAAGCAATAGATGGCTCTG CCAAGGACAGGTACGGCTGTCATC ggcatgaaagtTAAGCAATAGATGGCTCTG GTTGGCCAATCTACTCCC ggcatgaaagtTAAGCAATAGATGGCTCTG CCAAGGACAGGTACGGCTGTCATC tcacctagccccCTGCCGTTACTGCCCTG CTATTGGTCTCCTTAAACCTGTCTTG acctagcccTGCCGTTACTGCCCTG CTATTGGTCTCCTTAAACCTGTCTTG tcacctagccccAGCCATCTATTGCTTACATTT CCAGGGCCTCACCACCAAC gttcaccttgcccGGACAGGTACGGCTGTCATC CTATTGGTCTCCTTAAACCTGTCTTG ggccttaccaccGGTGAACGTGGATGAAGTT GAGAGTCAGTGCCTATCAG gccttaccaGGTGAACGTGGATGAAGTT CTATTGGTCTCCTTAAACCTGTCTTG ccaacccgcccaATGAAGTTGGTGGTGAGG GAGAGTCAGTGCCTATCAG aacccgcccATGAAGTTGGTGGTGAGG GAGAGTCAGTGCCTATCAG ccaacccgcccaATGAAGTTGGTGGTGAGG CTATTGGTCTCCTTAAACCTGTCTTG taccaaactgcccaATGAAGTTGGTGGTGAGG

Results c

Ts (°C) d

DPM

Length (base)

Tm

Orientation

12% gel

15% gel

20% gel

n

14

42

a

HT(−)

HT(−)

HT(−)

n

14

42

a

HT(−)

HT(−)

HT(−)

n

12

36

a

HT(−)

HT(±)

HT(±)

40

n

11

32

a

HT(−)

HT(+)

HT(+)

33

n

11

32

a

HT(−)

HT(±)

HT(±)

33

n

14

40

a

HT(−)

HT(−)

HT(−)

n

12

34

a

HT(−)

HT(±)

HT(±)

35

n

14

42

a

HT(−)

HT(±)

HT(±)

42

n

14

42

a

HT(−)

HT(−)

HT(−)

n

11

32

a

HT(−)

HT(+)

HT(+)

33

n

11

32

a

HT(−)

HT(±)

HT(±)

33

n

12

40

s

9

26

s

f

12

40

s

f

13

42

s

n

12

40

s

HT(+) HO(+) HT(+) HO(+) HT(−) HO(−) HT(−) HO(−) HT(±)

HT(+) HO(+) HT(+) HO(+) HT(−) HO(−) HT(−) HO(−) HT(+)

40

n

HT(−) HO(−) HT(−) HO(−) HT(−) HO(−) HT(−) HO(−) HT(−)

n

9

28

s

HT(−)

HT(+)

HT(+)

28

n

12

42

s

HT(−)

HT(±)

HT(+)

59

n

9

32

s

HT(−)

HT(+)

ut

52

n

12

42

s

HT(−)

HT(−)

HT(±)

55

n

14

42

s

HT(−)

HT(−)

HT(+)

52

28

W. Li et al. / Clinical Biochemistry 39 (2006) 833–842

1

IMH probe b

59

835

(continued on next page)

836

Table 1 (continued) Primers a

PCR fragments No

Length (bp)

Mutations checked

84

IVS-I-1 (G–T)

23

136

IVS-I-1 (G–T)

24

117

CDs41–42 (–TTCT)

25

72

CDs41–42 (–TTCT)

26

92

CDs41–42 (–TTCT)

27

113

CDs41–42 (–TTCT)

28

68

CDs41–42 (–TTCT)

29

117

CDs41–42 (–TTCT)

30

136

CD43 (G–T)

31

130

CD43 (G–T)

32

84

CD43 (G–T)

33

145

CDs71–72 (+A)

34

140

CDs71–72 (+A)

35

146

CD95 (+A)

36

144

CD95 (+A)

37

135

CD95 (+A)

38

132

CD95 (+A)

39

77

CD95 (+A)

40

144

IVS-II-654 (C–T)

Name

Sequences (5′→3′)

26R 1M β50 1M1 26R 41–42M β51 41–42M β3 41–42M1 β2 41–42M1 β51 41–42M1 β3 41–42N β51 43M β51 43M1 β51 43M1 β3 71–72M 71–72R 71–72M1 71–72R 95M β8 95M1 β8 95M2 95F 95M3 95F 95M1 71–72R 654M βC

GAGAGTCAGTGCCTATCAG taccaaactgcccaATGAAGTTGGTGGTGAGG CTATTGGTCTCCTTAAACCTGTCTTG ccaaactgcATGAAGTTGGTGGTGAGG GAGAGTCAGTGCCTATCAG cagaggttgagtcGTGGACAGATCCCCAAAG TAGGCACTGACTCTCTCTGCCTATT cagaggttgagtcGTGGACAGATCCCCAAAG CTGCTGGTGGTCTACCCTTGG aggttgagtGTGGACAGATCCCCAAAG TTGGTCTATTTTCCCACCCTTAG aggttgagtGTGGACAGATCCCCAAAG TAGGCACTGACTCTCTCTGCCTATT aggttgagtGTGGACAGATCCCCAAAG CTGCTGGTGGTCTACCCTTGG gaggttctttgaggGTGGACAGATCCCCAAAG TAGGCACTGACTCTCTCTGCCTATT gttcttttagtccttATAACAGCATCAGGAGTGGA TAGGCACTGACTCTCTCTGCCTATT cttttagtcATAACAGCATCAGGAGTGGA TAGGCACTGACTCTCTCTGCCTATT cttttagtcATAACAGCATCAGGAGTGGA CTGCTGGTGGTCTACCCTTGG catcacttaaaggcCTCATGGCAAGAAAGTGCT CACCCTGAAGTTCTCAGGA cacttaaaCTCATGGCAAGAAAGTGCT CACCCTGAAGTTCTCAGGA gcagctttgtcacaCCTTTGCCACACTGAGTG CCTTCCTATGACATGAACTTAACCAT cagctttgtcacCCTTTGCCACACTGAGTG CCTTCCTATGACATGAACTTAACCAT tgacaaagctgcTGAAGTTCTCAGGATCCACG ATGGCAAGAAAGTGCTCGGTG acaaagctgTGAAGTTCTCAGGATCCACG ATGGCAAGAAAGTGCTCGGTG cagctttgtcacCCTTTGCCACACTGAGTG CACCCTGAAGTTCTCAGGA gttaaggtaatagcaaGAAATATTTATATGCAGAAATA TTTCCCTAATCTCTTTCTTTCAGG

Results c

Ts (°C) d

DPM

Length (base)

Tm

Orientation

12% gel

15% gel

20% gel

n

14

42

s

HT(−)

HT(−)

HT(−)

n

9

28

s

HT(−)

HT(+)

ut

28

n

13

40

a

13

40

a

n

9

26

a

n

9

26

a

n

9

26

a

n

14

42

a

n

15

40

a

HT(+) HO(−) HT(−) HO(−) HT(+) HO(+) HT(+) HO(±) HT(±) HO(−) HT(+) HO(−) HT(−)

HT(+) HO(−) HT(±) HO(−) HT(+) HO(+) HT(+) HO(±) HT(±) HO(−) HT(+) HO(−) HT(+)

42

n

HT(+) HO(−) HT(+) HO(−) HT(+) HO(−) HT(+) HO(−) HT(−) HO(−) HT(+) HO(−) HT(−)

n

9

24

a

HT(−)

HT(+)

ut

28

n

9

24

a

HT(−)

HT(−)

HT(−)

n

14

40

s

HT(−)

HT(±)

HT(+)

42

n

8

20

s

HT(−)

HT(+)

HT(+)

28

n

14

42

s

HT(−)

HT(−)

HT(−)

n

12

36

s

HT(−)

HT(−)

HT(−)

n

12

36

a

HT(−)

HT(−)

HT(±)

42

n

9

26

a

HT(−)

HT(+)

ut

28

n

12

36

s

HT(−)

HT(−)

HT(−)

n

16

42

a

HT(−)

HT(−)

HT(−)

42 52 28 28 52 42 42

W. Li et al. / Clinical Biochemistry 39 (2006) 833–842

22

IMH probe b

103

IVS-II-654 (C–T)

42

140

IVS-II-654 (C–T)

43

99

IVS-II-654 (C–T)

44

138

IVS-II-654 (C–T)

45

97

IVS-II-654 (C–T)

46

136

IVS-II-654 (C–T)

47

95

IVS-II-654 (C–T)

48

68

IVS-II-654 (C–T)

49

125

IVS-II-654 (C–T)

50

121

IVS-II-654 (C–T)

51

118

IVS-II-654 (C–T)

52

68

IVS-II-654 (C–T)

53

161

Hb Constant Spring

54

157

Hb Constant Spring

55

178

Hb Quong Sze

56

172

Hb Quong Sze

57

115

Hb Quong Sze

a

654M β9 654M1 βC 654M1 β9 654M2 βC 654M2 β9 654M3 βC 654M3 β9 654M3 654F 654M4 654R 654M5 654R 654M6 654R 654M6 654R1 CSM αB CSM1 αB QSM α7 QSM1 α7 QSM1 QSR

gttaaggtaatagcaaGAAATATTTATATGCAGAAATA TATCATGCCTCTTTGCACCATTC ttaaggtaatagGAAATATTTATATGCAGAAATA TTTCCCTAATCTCTTTCTTTCAGG ttaaggtaatagGAAATATTTATATGCAGAAATA TATCATGCCTCTTTGCACCATTC taaggtaatagAAATATTTATATGCAGAAATATTG TTTCCCTAATCTCTTTCTTTCAGG taaggtaatagAAATATTTATATGCAGAAATATTG TATCATGCCTCTTTGCACCATTC aaggtaataAAATATTTATATGCAGAAATATTG TTTCCCTAATCTCTTTCTTTCAGG aaggtaataAAATATTTATATGCAGAAATATTG TATCATGCCTCTTTGCACCATTC aaggtaataAAATATTTATATGCAGAAATATTG GAATAACAGTGATAATTTCTGG ttgctattaccttaacGAATAACAGTGATAATTTCTGG CTGCTATTAGCAATATGAAAC gctattaccttaGAATAACAGTGATAATTTCTGG CTGCTATTAGCAATATGAAAC tattaccttGAATAACAGTGATAATTTCTGG CTGCTATTAGCAATATGAAAC tattaccttGAATAACAGTGATAATTTCTGG GAATATTTTATATGCAGAAATATTG ccagcttgacggtaGAGCACCGTGCTGACCTCCAAA CAGGCTGCCGCCCACTCAGAC gcttgacggGAGCACCGTGCTGACCTCCAAA CAGGCTGCCGCCCACTCAGAC ttgtccggggaggGCCGAGTTCACCCCTGCGGTG CAGGAAGGGCCGGTGCAAGG tccggggCCGAGTTCACCCCTGCGGTG CAGGAAGGGCCGGTGCAAGG tccggggCCGAGTTCACCCCTGCGGTG GGAACGGCTACCGAGGCTCC

n

16

42

a

HT(−)

HT(−)

HT(−)

n

12

30

a

HT(−)

HT(−)

HT(±)

n

12

30

a

HT(−)

HT(−)

HT(−)

n

11

28

a

HT(−)

HT(±)

HT(±)

n

11

28

a

HT(−)

HT(−)

HT(−)

n

9

22

a

HT(−)

HT(+)

HT(+)

n

9

22

a

HT(−)

HT(−)

HT(−)

n

9

22

a

HT(−)

HT(−)

HT(−)

n

16

38

s

HT(−)

HT(−)

HT(−)

n

12

32

s

HT(−)

ut

HT(±)

32

n

9

22

s

HT(−)

HT(±)

HT(±)

22

n

9

22

s

HT(−)

HT(−)

HT(±)

22

n

14

42

s

9

30

s

n

13

44

s

n

7

26

s

n

7

26

s

HT(−) HO(±) HT(+) HO(+) HT(−) HO(−) HT(±) HO(±) HT(+) HO(+)

HT(+) HO(+) HT(+) HO(+) HT(+) HO(−) HT(−) HO(−) ut ut

40

n

HT(−) HO(−) HT(−) HO(−) HT(−) HO(−) HT(−) HO(−) HT(−) HT(−)

28

26

33 59 40 40

The sequences written in small letter are the sequences of IMH probes. The bold and underlined letters are the mutation sites. DPM represents the distance between snapback PCR primers and mutation sites. f = far away from the mutation; n = near by the mutation. b Tm is calculated by using Wallace's formula, Tm = 2(A + T) + 4(C + G). Letter ‘a’ denotes antisense strand probes. Letter ‘s’ denotes sense strand probes. c HT = heterozygote; HO = homozygote; ut = untested. (+) and (−) denoted that the mutation could be detected and not be detected, respectively; (±) denotes that the resolution of snapback SSCP was bad though the mutation could be detected. d Ts = the optimal temperature of snapback SSCP. e Denotes the external primers of nested PCR.

W. Li et al. / Clinical Biochemistry 39 (2006) 833–842

41

837

838

W. Li et al. / Clinical Biochemistry 39 (2006) 833–842

Fig. 1. Schematic diagrams of IMH and the location of PCR primers which were listed in Table 2. The arrows represent PCR primers. (A) (1) DNA conformation with IMH; (2) DNA conformation without IMH. (B) The location of β-thalassemia point mutations and PCR primers. (C) The location of α-thalassemia point mutations and PCR primers.

acetate acid. Loading dye solution contained 10% sucrose and 0.05% bromophenol blue (BPB). 1–4 μL of the second round PCR products (containing about 50 ng of DNA) was added into 3 μL of loading dye solution and denatured at 98°C for 30 s, then chilled on ice to generate DNA single strand. The electrophoretic unit (Hoefer SE 260, Amersham Bioscience, USA) was placed in an incubator (MIR-153, SANYO, Japan, when the electrophoretic temperature was ≤52°C, or PYXDHS.500, Shanghai Yuejin Medical instrument CO., LDT, China, when the temperature was > 52°C) to control the electrophoretic temperature. Tank buffer was pre-cooled or pre-heated by using refrigerator or microwave oven before electrophoresis to keep the temperature of tank buffer consistent with the electrophoretic temperature as possible. Electrophoreses were run at 300 V for 1–2 h. Electrophoretic temperatures from 15 to 62°C were tested to find out the

optimal temperature for each snapback SSCP fragment. Once a mutation was identified by using a snapback SSCP fragment, we would repeat snapback SSCP at least 4 times under the same conditions by using the same snapback SSCP fragment generated by different PCRs. Silver stain The gel was stained by using Bassam's method [12] after electrophoresis was over. Some modifications were made to save time. Briefly, fix a mini-gel with 50 mL of 10% acetate acid for 5 min and then wash with 150 mL of deionized water, 3 times (3 min/time). Stain the gel with 20 mL of 0.1% AgNO3 containing 30 μL of 37% formaldehyde for 5 min. Reveal the gel with 20 mL of 1.5% NaCO3 containing 30 μL of 37% formaldehyde and 20 μL of 2 mg/mL Na2S2O3 for 2 to 3 min

W. Li et al. / Clinical Biochemistry 39 (2006) 833–842

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Fig. 2. DNA band patterns of snapback SSCP for 13 types of known thalassemia mutations. 15% PAGE at 300 V for 1.5 h. The arrows indicate the abnormal bands. The sequences of PCR primers are listed in Table 2. (A) Primers: − 28M2 and β1. Lanes 1, 2, and 3: heterozygote of −28 (A→G), heterozygote of − 29 (A→G) and the normal control. (B) Primers: − 29M1 and β1. Lanes 1, 2, and 3: heterozygote of − 28 (A→G), heterozygote of − 29 (A→G) and the normal control. (C) Primers: 17M1 and β50. Lanes 1, 2, and 3: homozygote of codon 17 (A→T), heterozygote of codon 17 (A→T) and the normal control. (D) Primers: 26M1 and β50. Lanes 1, 2, 3, and 4: heterozygote of codon 26 (G→A, HbE), heterozygote of codon 30 (A→G), heterozygote of IVS-I-1 (G→T) and the normal control. (E) Primers: 30M1 and 26R. Lanes 1, 2, and 3: heterozygote of codon 30 (A→G), the normal control and heterozygote of IVS-I-1 (G→T). (F) Primers: 1M1 and 26R. Lanes 1, 2, and 3: heterozygote of IVS-I-1 (G→T), heterozygote of codon 30 (A→G) and the normal control. (G) Primers: 41–42M1 and β2. Lanes 1, 2, and 3: homozygote of codons 41–42 (–TTCT), heterozygote of codons 41–42 (–TTCT) and the normal control. (H) Primers: 43M1 and β51. Lanes 1, 2, and 3: heterozygote of codons 41–42 (–TTCT), heterozygote of codon 43 (G→T) and the normal controls. (I) Primers: 71–72M1 and 71–72R. Lanes 1 and 2: heterozygote of codons 71–72 (+A) and the normal control. (J) Primers: 95M3 and 95F. Lanes 1 and 2: heterozygote of codon 95 (+ A) and the normal control. (K) Primers: 654M3 and βC. Lanes 1 and 2: heterozygote of IVS-II-654 (C→T) and the normal control. (L) Primer: CSM1 and αB. Lanes 1, 2, and 3: the normal control, heterozygote of Hb Constant Spring and double heterozygote for Hb Constant Spring and --sea. (M) Primers: QSM1 and QSR. Lanes 1, 2, and 3: the normal control, heterozygote of Hb Quong Sze and double heterozygote for Hb Quong Sze and --sea.

until DNA bands appeared clearly, and finally, stop silver stain with 50 mL of 10% acetate acid. Results The results of snapback SSCP for all 57 snapback SSCP fragments are summarized in Table 1. Fig. 2 shows that all specimens (including heterozygotes for 13 thalassemia point mutations, homozygotes for codon 17 (A→T) and codons 41–42 (–TTCT), double heterozygote for Hb Constant Spring and --sea, and double heterozygote for Hb Quong Sze and --sea) could be identified by using 15% SSCP gel and 13 different IMH probes which were 7–11 bases long. These 13 IMH probes were chosen from Table 1 and listed in Table 2 to make our description pellucid. There were both normal and abnormal DNA bands in the heterozygote lanes, but only the abnormal bands were present in the homozygote lane. There were no abnormal bands in the normal controls lanes. The DNA band patterns of the mutations which were complementary with IMH probes were obviously

different from those of the mutations which were not complementary with IMH probe (Figs. 2A, B, D, E, F, and H). The results of snapback SSCP using IMH probes given in Table 2 were completely consistent with those of PCR-ASO. No false negative or false positive DNA band patterns were found for those IMH probes listed in Table 2 under the optimal conditions. The same results were obtained under the same conditions when we repeated snapback SSCP with the same snapback SSCP fragment generated by different PCRs. The reproducibility of snapback SSCP was 100%. Table 1 shows that sensitivity and resolution of snapback SSCP were impacted by several factors such as gel concentration, electrophoretic temperature, locations of PCR primers, and lengths of snapback SSCP fragments and IMH probes, etc. Most mutations could not be identified in 12% SSCP gels, but the heterozygote of codon 41–42(–TTCT) could be identified in a 12–20% SSCP gel because the migration rate of the DNA single strand was determined by the length of the single-strand DNA when the latter's conformation was unfolded by heat. The

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Table 2 13 snapback SSCP fragments which worked well in 15% PAGE Fragments

Length (bp)

Mutation

1

93

− 28 (A–G)

2

93

− 29 (A–G)

3

113

CD17 (A–T)

4

90

CD26 (G–A)

5

136

CD30 (A–G)

6

136

IVS-I-1 (G–T)

7

92

8

130

CD43 (G–T)

9

140

CDs71–72 (+A)

10

132

CD95 (+A)

11

136

IVS-II-654 (C–T)

12

157

Hb Constant Spring

13

115

Hb Quong Sze

CDs41–42 (–TTCT)

Primers

Tm (°C)

Ts (°C)

Name

Sequences (5′→3′)

Probe

Primer

− 28M2 β1 − 29M1 β1 17M1 β50 26M1 β50 30M1 26R 1M1 26R 41–42M1 β2 43M1 β51 71–72M1 71–72R 95M3 95F 654M3 βC CSM1 αB QSM1 QSR

gcatagaagtcTAAGCAATAGATGGCTCTG GTTGGCCAATCTACTCCC ggcatgaaagtTAAGCAATAGATGGCTCTG GTTGGCCAATCTACTCCC acctagccCTGCCGTTACTGCCCTG CTATTGGTCTCCTTAAACCTGTCTTG gccttaccaGGTGAACGTGGATGAAGTT CTATTGGTCTCCTTAAACCTGTCTTG aacccgcccATGAAGTTGGTGGTGAGG GAGAGTCAGTGCCTATCAG ccaaactgcATGAAGTTGGTGGTGAGG GAGAGTCAGTGCCTATCAG aggttgagtGTGGACAGATCCCCAAAG TTGGTCTATTTTCCCACCCTTAG cttttagtcATAACAGCATCAGGAGTGGA TAGGCACTGACTCTCTCTGCCTATT cacttaaaCTCATGGCAAGAAAGTGCT CACCCTGAAGTTCTCAGGA acaaagctgTGAAGTTCTCAGGATCCACG ATGGCAAGAAAGTGCTCGGTG aaggtaataAAATATTTATATGCAGAAATATTG TTTCCCTAATCTCTTTCTTTCAGG gcttgacggGAGCACCGTGCTGACCTCCAAA CAGGCTGCCGCCCACTCAGAC tccggggCCGAGTTCACCCCTGCGGTG GGAACGGCTACCGAGGCTCC

32

54 56 54 56 56 74 56 74 54 58 54 58 56 66 58 72 56 58 64 64 56 66 70 72 72 68

32 26 28 32 28 26 24 20 26 22 30 26

33 33 28 28 52 28 52 28 28 28 26 33 40

Tm is calculated with Wallace's formula. Ts = the optimal temperature of snapback SSCP. The sequences written in small letter are the sequences of IMH probes. The bold and underlined letters are the mutation sites.

resolution of snapback SSCP using a 20% gel was better than that using a 15% gel, given that the IMH probes used were ≥ 12 bases, but the resolution using a 15% gel was better than that using a 20% gel if the IMH probes used were 7–9 bases long. Every snapback SSCP fragment had its own optimal electrophoretic temperature which was over or around the melting temperature (Tm) of its corresponding IMH probe. The sensitivity of snapback SSCP would decrease when the electrophoretic temperature was beyond the working temperature range of each snapback SSCP fragment. When snapback SSCP fragments were too long or too short, some mutations could not be identified. For example, codon 43 (G→T), codon 95 (+ A) and IVS-II-654(C→T) could not be identified by using snapback SSCP fragments which were <90 bp. The optimal length of snapback SSCP fragments for 15%–20% SSCP gels was 90–180 bp. Codon 17 (A→T) could not be identified when the snapback PCR primers were placed far away from the mutation site. The heterozygotes of some mutations (such as − 28 (A→G), − 29 (A→G) and Hb Quong Sze, etc) could not be identified by using 14–15 bases long IMH probes, whereas the double heterozygote for Hb Quong Sze and --sea which was equal to the homozygote of Hb Quong Sze could be identified well by using the same probe as used for heterozygote. Mutations could be detected by using either sense strand probes or antisense strand probe. However, sometimes the direction of IMH probe could impact the sensitivity or resolution of

snapback SSCP. For example, the resolution of the sense strand probe was worse than that of the antisense strand probe for codon 95 (+ A). Discussion We have detected 13 types of known thalassemia point mutations by using snapback SSCP under various conditions. All specimens (including heterozygotes of 13 thalassemia point mutations, homozygotes of codon 17 (A→T) and codons 41–42 (–TTCT), double heterozygote for Hb Constant Spring and --sea, and double heterozygote for Hb Quong Sze and --sea) could be identified by using 15% SSCP gel and 7–11 bases-long IMH probes (Fig. 2 and Table 2). The advantages of snapback SSCP are (1) it is simple as compared to PCR-ASO; (2) snapback SSCP is specific and stable once the conditions of snapback SSCP are optimized. The activities and specificity of restriction endonucleases do not cause any interference as no enzyme is used after PCR; (3) samples can be checked at any time without the limit of half-life of radioactive isotope; and (4) since PCR products used in snapback SSCP can be checked by using 2% agarose gel before snapback SSCP, misdiagnoses caused by false positive or false negative PCR can be reduced significantly in snapback SSCP. All these advantages of snapback SSCP are important especially for prenatal diagnosis. The differences between snapback SSCP and ordinary SSCP are (1) snapback

W. Li et al. / Clinical Biochemistry 39 (2006) 833–842

SSCP is time-saving (only about 1.5 h for 15% gel); (2) the optimal temperatures of ordinary SSCP are intrinsic and able to be estimated by our formula [11], while the optimal temperatures of snapback SSCP are usually over or around Tm of IMH probes. Therefore, it is possible to adjust the optimal temperature of snapback SSCP by changing the lengths of IMH probes; (3) since DNA conformation of snapback SSCP is formed by the specific hybridization between two complementary sequences, it is more heat-stable than that of ordinary SSCP. Therefore, the temperature of ordinary SSCP has to be controlled more strictly than that of snapback SSCP. Direct DNA sequencing is considered the gold standard for mutation detection, but it is much more expensive than snapback SSCP. The cost of direct DNA sequencing is about 18 US dollars/test including the cost of PCR, purification of PCR products and bidirectional sequencing, whereas the cost of snapback SSCP is about only 0.44 US dollars/test including the cost of two round PCR, gel electrophoresis, and silver stain. Since an automatic sequencer is so expensive that many hospitals can not afford it, samples from these hospitals have to be sent elsewhere for sequencing, which usually takes around 5 to 7 days and hence does not favor diagnosis of diseases. To screen 13 types of thalassemia mutations for a patient by using snapback SSCP, we have to perform 15 PCR reactions including 2 first rounds PCRs and 13 second round PCRs using the primers listed in Table 2. If these 13 thalassemia mutations are going to be examined by direct DNA sequencing, 4 PCR fragments have to be amplified and used as sequencing templates. One of them is from the α-globin gene, and the other 3 are from the β-globin gene. These PCR products have to be purified before sequencing. The time consumed during postPCR handling of samples such as purification of PCR products and sequencing reactions required by direct sequencing are equivalent to that consumed by 3 PCR reactions. Therefore, direct DNA sequencing can save about 20% time as compared to snapback SSCP. However, considering that bidirectional sequencing spends about 18 US dollars, while snapback SSCP spends only 0.44 US dollars, we think that snapback SSCP is more cost-effective than direct DNA sequencing. In addition, the frequency of every thalassemia mutation is different from each other in a population or a region. For example, in Guangxi, a province in south China, codons 41–42 (–TTCT) and codon 17 (A→T) are the most common β-thalassemia point mutations. Both of them account for about 70% of the β-thalassemia point mutations. The next are IVS-II-654(C→T) and −28 (A→G) mutations. Both of them account for about 18.6%. The rest are the rare mutations, accounting for only about 11.4%. Therefore, we can identify a thalassemia mutation for a patient usually by using only 1–4 snapback PCR primer pair(s). In that case, direct DNA sequencing has no obvious dominance as compared to snapback SSCP. Other authors also consider that DNA sequencing was expensive and labour-intensive [13,14]. We think the advantages of direct DNA sequencing are that unknown point mutations must be characterized by using direct DNA sequencing and that rare mutations can be identified more quickly by using direct DNA sequencing than using snapback SSCP when the mutation background of a patient is unknown.

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We could not compare snapback SSCP with DGGE or DHPLC because we do not have experience in DGGE and DHPLC. The major disadvantage of snapback SSCP is that its sensitivity and resolution are impacted by the location of snapback PCR primer, the length of snapback SSCP fragment and IMH probe, and electrophoretic temperature, etc. Therefore, the conditions of snapback SSCP must be optimized. We find that 9 bases-long IMH probes are suitable for most mutations, while 7–8 bases long IMH probes are suitable for those mutations (such as Hb Quong Sze, etc.) locating in CG-rich sequences. According to what have been shown in Table 1, the conditions of snapback SSCP could be optimized by following these principles: (a) select appropriate concentration of PAG and length of IMH probe. 15% gel and 9 bases long IMH probes should be the first choice for most mutations; (b) limit the length of snapback SSCP fragments between 90–180 bp for 15% gel; (c) set snapback PCR primers near the mutation site; (d) change the direction of IMH probe or change the location of PCR primers; and (e) run electrophoresis at the optimal snapback SSCP temperature which is usually over or around Tm of the IMH probe. Application of these principles could make it easy to optimize the conditions of snapback SSCP for most known thalassemia mutations. Conclusion Snapback SSCP can be applied to the detection of known thalassemia point mutations, but the conditions of snapback SSCP must be optimized. Once these conditions are optimized, snapback SSCP is cost-effective, specific and stable. 15% gel and 9 bases-long IMH probes should be the first choice for most mutations. Acknowledgments We are very grateful to Dr. Y. Hattori and Professor Ku. Yamamoto of Yamaguchi University School of Medicine, Japan, for their kind help in SSCP in 1994. References [1] Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified β-globin and HLA-DQ alpha DNA with allelespecific oligonucleotide probes. Nature 1986;324:163–6. [2] Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A 1989;86(16):6230–4. [3] Newton CR, Graham A, Heptinstall LE, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989;17:2503–16. [4] Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci U S A 1991;88:189–93. [5] Pirastu M, Ristaldi MS, Cao A. Prenatal diagnosis of beta thalassemia based on restriction endonuclease analysis of amplified fetal DNA. J Med Genet 1989;26:363–7. [6] Drobyshev A, Mologina N, Shik V, Pobedimskaya D, Yershov G, Mirzabekov A. Sequence analysis by hybridization with oligonucleotide microchip: identification of beta-thalassemia mutations. Gene 1997;188: 45–52. [7] Losekoot M, Fodde R, Harteveld CL, van Heeren H, Giordano PC, Bernini

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LF. Denaturing gradient gel electrophoresis and direct sequencing of PCR amplified genomic DNA: a rapid and reliable diagnostic approach to beta thalassaemia. Br J Haematol 1990;76:269–74. [8] Huber CG, Premstaller A, Xiao W, Oberacher H, Bonn GK, Oefner PJ. Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns. J Biochem Biophys Methods 2001;47:5–19. [9] Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A 1989;86:2766–70. [10] Wilton SD, Honeyman K, Fletcher S, Laing NG. Snapback SSCP analysis: engineered conformation changes for the rapid typing of known mutations. Hum Mutat 1998;11:252–68.

[11] Li W, Gao F, Liang JL, Li CS, et al. Estimation of the optimal electrophoretic temperature of DNA single strand conformation by DNA base composition. Electrophoresis 2003;24:2283–9. [12] Bassam BJ, Caetano-Anolles G, Gresshoff PM. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 1991;196:80–3. [13] Boutin P, Vasseur F, Samson C, Wahl C, Froguel P. Routine mutation screening of HNF-1alpha and GCK genes in MODY diagnosis: how effective are the techniques of DHPLC and direct sequencing used in combination? Diabetologia 2001;44:775–8. [14] Mogensen J, Bahl A, Kubo T, Elanko N, Taylor R, McKenna WJ. Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy. J Med Genet 2003;40:e59.