J Clin Med Res

Journal of Clinical Medicine Research, ISSN 1918-3003 print, 1918-3011 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Med Res and Elmer Press Inc

Journal website http://www.jocmr.org

Original Article

Volume 10, Number 7, July 2018, pages 552-561

Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in Hypervariable Regions

**Table 1.** PCR Primer Sequences
Target	Forward	Reverse	Amplicon size (bp)
SNP and deletion [30]
3010G>A	2698	AGAGGCGGGCATAACACAGCA	3066	GATCACGTAGGACTTTAATCGTTGA	369
4386T>C	4344	TCGAACCCATCCCTGAGAATCC	4577	GTTTATTTCTAGGCCTACTCAGGTAA	234
5178C>A	4989	CAGCTACGCAAAATCTTAGCATAC	5257	TTGGGCAAAAAGCCGGTTAGCG	269
8272C>del	8153	GGGGTATACTACGGTCAATGCTC	8530	TCATTTTGGTTCTCAGGGTTTGTTAT	378
8794C>T	8628	CAAATATCTCATCAACAACCGACTA	8994	CAGGGCTATTGGTTGAATGAGTAG	367
10397A>G	10277	ACCCCTACCATGAGCCCTACAA	10515	GTGAGATGGTAAATGCTAGTATAATAT	239
C-stretch [36]
HV1C	15896	CAAATGGGCCTGTCCTTGTA	16414	TGTGCGGGATATTGATTTC	519
HV2C	29	GGTCTATCACCCTATTAACCAC	408	CTGTTAAAAGTGCATACCGCCA	380
Others
ND1 [38]	3344	TTCTAATCGCAATGGCATTCCT	3452	AAGGGTTGTAGTAGCCCGTAG	109
D17Z1 [37]	126	TTTTGCAGGATCTACAAGTGGA	332	AAGAGGTCTACATGTCCCCTTG	207

Table 1. PCR Primer Sequences

Target

Forward

Reverse

Amplicon size (bp)

Position

Sequence (5′ to 3′)

Position

Sequence (5′ to 3′)

SNP and deletion [30]

3010G>A

2698

AGAGGCGGGCATAACACAGCA

3066

GATCACGTAGGACTTTAATCGTTGA

369

4386T>C

4344

TCGAACCCATCCCTGAGAATCC

4577

GTTTATTTCTAGGCCTACTCAGGTAA

234

5178C>A

4989

CAGCTACGCAAAATCTTAGCATAC

5257

TTGGGCAAAAAGCCGGTTAGCG

269

8272C>del

8153

GGGGTATACTACGGTCAATGCTC

8530

TCATTTTGGTTCTCAGGGTTTGTTAT

378

8794C>T

8628

CAAATATCTCATCAACAACCGACTA

8994

CAGGGCTATTGGTTGAATGAGTAG

367

10397A>G

10277

ACCCCTACCATGAGCCCTACAA

10515

GTGAGATGGTAAATGCTAGTATAATAT

239

C-stretch [36]

HV1C

15896

CAAATGGGCCTGTCCTTGTA

16414

TGTGCGGGATATTGATTTC

519

HV2C

GGTCTATCACCCTATTAACCAC

408

CTGTTAAAAGTGCATACCGCCA

380

Others

ND1 [38]

3344

TTCTAATCGCAATGGCATTCCT

3452

AAGGGTTGTAGTAGCCCGTAG

109

D17Z1 [37]

126

TTTTGCAGGATCTACAAGTGGA

332

AAGAGGTCTACATGTCCCCTTG

207

**Table 2.** ASPE Primer Sequences for Detection of Five SNPs and One Deletion in MtDNA
	Sequence (5′ to 3′)
Sequences in parentheses indicate 24-mer TAG sequences.
3010G>A	(CTATCATTTATCTCTTTCTCAATT) ATGTTGGATCAGGACATCCCG
	(CAATAAACATTCTTTACATTCTCA) GATGTTGGATCAGGACATCCCA
4386T>C	(CTACAAACACTTAACTTTATCTTT) CCTTACTTTAGGATGGGGTGTGT
	(CATAATCAATTTCAACTTTCTACT) CTTACTTTAGGATGGGGTGTGC
5178C>A	(ATTAAACAACTCTTAACTACACAA) TATCTCGCACCTGAAACAAGC
	(ATACTTTACAAACAAATAACACAC) CTATCTCGCACCTGAAACAAGA
8272C>del	(AATTTCTTCTCTTTCTTTCACAAT) CCCTATAGCACCCCCTCTAC
	(CATCTTCATATCAATTCTCTTATT) CCCTATAGCACCCCCTCTAG
8794C>T	(TTAATACAATTCTCTCTTTCTCTA) TGGGTGGTTGGTGTAAATGAGTG
	(AACTTTCTCTCTCTATTCTTATTT) TGGGTGGTTGGTGTAAATGAGTA
10397A>G	(TTAACAACTTATACAAACACAAAC) TGACTACAAAAAGGATTAGACTGA
	(ATCTCAATTACAATAACACACAAA) TGACTACAAAAAGGATTAGACTGG

Table 2. ASPE Primer Sequences for Detection of Five SNPs and One Deletion in MtDNA

Sequence (5′ to 3′)

Sequences in parentheses indicate 24-mer TAG sequences.

3010G>A

(CTATCATTTATCTCTTTCTCAATT) ATGTTGGATCAGGACATCCCG

(CAATAAACATTCTTTACATTCTCA) GATGTTGGATCAGGACATCCCA

4386T>C

(CTACAAACACTTAACTTTATCTTT) CCTTACTTTAGGATGGGGTGTGT

(CATAATCAATTTCAACTTTCTACT) CTTACTTTAGGATGGGGTGTGC

5178C>A

(ATTAAACAACTCTTAACTACACAA) TATCTCGCACCTGAAACAAGC

(ATACTTTACAAACAAATAACACAC) CTATCTCGCACCTGAAACAAGA

8272C>del

(AATTTCTTCTCTTTCTTTCACAAT) CCCTATAGCACCCCCTCTAC

(CATCTTCATATCAATTCTCTTATT) CCCTATAGCACCCCCTCTAG

8794C>T

(TTAATACAATTCTCTCTTTCTCTA) TGGGTGGTTGGTGTAAATGAGTG

(AACTTTCTCTCTCTATTCTTATTT) TGGGTGGTTGGTGTAAATGAGTA

10397A>G

(TTAACAACTTATACAAACACAAAC) TGACTACAAAAAGGATTAGACTGA

(ATCTCAATTACAATAACACACAAA) TGACTACAAAAAGGATTAGACTGG

**Table 3.** ASPE Primer Sequences for Detection of HV1C Groups in MtDNA
Group	Sequence (5′ to 3′)
Sequences in parentheses indicate 24-mer TAG sequences. Position at 16194 is A.
0CT	(CTAAATCACATACTTAACAACAAA) TAAAAACCCAATCCACATCAAAAT
1CT	(ACAAATATCTAACTACTATCACAA) AAAAACCCAATCCACATCAAAACT
3CT	(TACTTAAACATACAAACTTACTCA) AAACCCAATCCACATCAAAACCCT
4CT	(AATCAACACACAATAACATTCATA) ACCCAATCCACATCAAAACCCCT
5CT	(CTAAACATACAAATACACATTTCA) CCAATCCACATCAAAACCCCCT
8CT	(CAAATACATAATCTTACATTCACT) CCACATCAAAACCCCCCCCT
9C	(TACACAAACAATCTTTCACAATTT) CACATCAAAACCCCCCCCCA
10C	(AATAACAACTCACTATATCATAAC) ACATCAAAACCCCCCCCCCA
11C	(CAAACAAACATTCAAATATCAATC) CATCAAAACCCCCCCCCCC

Table 3. ASPE Primer Sequences for Detection of HV1C Groups in MtDNA

Group

Sequence (5′ to 3′)

Sequences in parentheses indicate 24-mer TAG sequences. Position at 16194 is A.

0CT

(CTAAATCACATACTTAACAACAAA) TAAAAACCCAATCCACATCAAAAT

1CT

(ACAAATATCTAACTACTATCACAA) AAAAACCCAATCCACATCAAAACT

3CT

(TACTTAAACATACAAACTTACTCA) AAACCCAATCCACATCAAAACCCT

4CT

(AATCAACACACAATAACATTCATA) ACCCAATCCACATCAAAACCCCT

5CT

(CTAAACATACAAATACACATTTCA) CCAATCCACATCAAAACCCCCT

8CT

(CAAATACATAATCTTACATTCACT) CCACATCAAAACCCCCCCCT

(TACACAAACAATCTTTCACAATTT) CACATCAAAACCCCCCCCCA

10C

(AATAACAACTCACTATATCATAAC) ACATCAAAACCCCCCCCCCA

11C

(CAAACAAACATTCAAATATCAATC) CATCAAAACCCCCCCCCCC

**Table 4.** ASPE Primer Sequences for Detection of HV2C Groups in MtDNA
Group	Sequence (5′ to 3′)
Sequences in parentheses indicate 24-mer TAG sequences. Position at 316 is G.
6CT	(TCTCTTTAAACACATTCAACAATA) AAAAAATTTCCACCAAACCCCCCT
7CT	(CTTTCTTAATACATTACAACATAC) AAAATTTCCACCAAACCCCCCCT
8CT	(CAATTTACATTTCACTTTCTTATC) TTTCCACCAAACCCCCCCCT
9CT	(CATAAATCTTCTCATTCTAACAAA) CCACCAAACCCCCCCCCT
11C	(TACAACATCTCATTAACATATACA) ACCAAACCCCCCCCCCCG
13C	(AATCTCTACAATTTCTCTCTAATA) ACCAAACCCCCCCCCCCC

Table 4. ASPE Primer Sequences for Detection of HV2C Groups in MtDNA

Group

Sequence (5′ to 3′)

Sequences in parentheses indicate 24-mer TAG sequences. Position at 316 is G.

6CT

(TCTCTTTAAACACATTCAACAATA) AAAAAATTTCCACCAAACCCCCCT

7CT

(CTTTCTTAATACATTACAACATAC) AAAATTTCCACCAAACCCCCCCT

8CT

(CAATTTACATTTCACTTTCTTATC) TTTCCACCAAACCCCCCCCT

9CT

(CATAAATCTTCTCATTCTAACAAA) CCACCAAACCCCCCCCCT

11C

(TACAACATCTCATTAACATATACA) ACCAAACCCCCCCCCCCG

13C

(AATCTCTACAATTTCTCTCTAATA) ACCAAACCCCCCCCCCCC

Table 5. Frequency of Type Consisting of HV1C Pattern, HV2C Pattern, and Major Haplogroups for Japanese Populations

HV1C

HV2C

M7a

Others

Frequency N (%)

0CT

8CT

1 (0.7)

3CT

7CT

3 (2.0)

8CT

4 (2.7)

8CT>9CT

1 (0.7)

5CT

7CT

1 (0.7)

8CT

2 (1.3)

Others

1 (0.7)

10C

7CT

1 (0.7)

11C

8CT>7CT

1 (0.7)

13C>11C

1 (0.7)

3CT>4CT

9CT>8CT

1 (0.7)

5CT>8CT

7CT

1 (0.7)

5CT>10C

7CT

30 (20.0)

8CT

29 (19.3)

7CT>8CT

1 (0.7)

8CT>7CT

7 (4.7)

8CT>9CT

2 (1.3)

9CT>8CT

2 (1.3)

Others

2 (1.3)

10C>5CT

8CT

2 (1.3)

8CT>7CT

1 (0.7)

10C>11C

6CT

1 (0.7)

8CT

3 (2.0)

Others

4 (2.7)

5CT>10C>9C

7CT

3 (2.0)

8CT>7CT

3 (2.0)

8CT>9CT

1 (0.7)

10C>3CT>9C

8CT

1 (0.7)

10C>11C>9C

7CT

13 (8.7)

8CT

12 (8.0)

6CT>7CT

1 (0.7)

7CT>13C

1 (0.7)

8CT>7CT

1 (0.7)

Others

5 (3.3)

11C>10C>9C

7CT

1 (0.7)

8CT

1 (0.7)

13C>9CT

1 (0.7)

Others

1 (0.7)

Others

7CT

2 (1.3)

8CT

1 (0.7)

150

7.3

10.7

37.3

3.3

9.3

32.0

**Table 6.** Frequency of HV1C Pattern in the Major Haplogroups for Japanese Populations
HV1C	A	B	D4	D5	M7a	Others	Frequency N (%)
0CT						1	1 (0.7)
3CT	7		1				8 (5.3)
5CT			3			1	4 (2.7)
10C						1	1 (0.7)
11C		1				1	2 (1.3)
3CT>4CT						1	1 (0.7)
5CT>8CT			1				1 (0.7)
5CT>10C	3		43		10	17	73 (48.7)
10C>5CT			1			2	3 (2.0)
10C>11C		3		2		3	8 (5.3)
5CT>10C>9C			3		3	1	7 (4.7)
10C>3CT>9C	1						1 (0.7)
10C>11C>9C		10	4	2	1	16	33 (22.0)
11C>10C>9C		2				2	4 (2.7)
Others				1		2	3 (2.0)

Table 6. Frequency of HV1C Pattern in the Major Haplogroups for Japanese Populations

HV1C

M7a

Others

Frequency N (%)

0CT

1 (0.7)

3CT

8 (5.3)

5CT

4 (2.7)

10C

1 (0.7)

11C

2 (1.3)

3CT>4CT

1 (0.7)

5CT>8CT

1 (0.7)

5CT>10C

73 (48.7)

10C>5CT

3 (2.0)

10C>11C

8 (5.3)

5CT>10C>9C

7 (4.7)

10C>3CT>9C

1 (0.7)

10C>11C>9C

33 (22.0)

11C>10C>9C

4 (2.7)

Others

3 (2.0)

**Table 7.** Frequency of HV2C Pattern in the Major Haplogroups for Japanese Populations
HV2C	A	B	D4	D5	M7a	Others	Frequency N (%)
6CT				1			1 (0.7)
7CT	2	2	25		6	20	55 (36.7)
8CT	7	7	19	3	2	18	56 (37.3)
6CT>7CT				1			1 (0.7)
7CT>8CT	1						1 (0.7)
8CT>7CT			8		3	2	13 (8.7)
7CT>13C		1					1 (0.7)
8CT>9CT	1		1		2		4 (2.7)
9CT>8CT			1			2	3 (2.0)
13C>9CT						1	1 (0.7)
13C>11C		1					1 (0.7)
Others		5	2		1	5	13 (8.7)

Table 7. Frequency of HV2C Pattern in the Major Haplogroups for Japanese Populations

HV2C

M7a

Others

Frequency N (%)

6CT

1 (0.7)

7CT

55 (36.7)

8CT

56 (37.3)

6CT>7CT

1 (0.7)

7CT>8CT

1 (0.7)

8CT>7CT

13 (8.7)

7CT>13C

1 (0.7)

8CT>9CT

4 (2.7)

9CT>8CT

3 (2.0)

13C>9CT

1 (0.7)

13C>11C

1 (0.7)

Others

13 (8.7)

**Table 8.** Differences in C-stretch Patterns Between 13 Pairs of Urine and Blood Samples
N	HV1C	HV2C
1	0CT>5CT	0CT
2	5CT>10C	5CT
1	5CT>10C	5CT>10C>9C	8CT	8CT>7CT
1	10C>11C	10C>11C>9C
1	10C>11C>9C	10C>11C
1	10C>11C>9C	10C>11C	6CT>7CT	6CT
1	11C	Others
1			7CT	7CT>13C
2			8CT	8CT>7CT
1			8CT>7CT	8CT
1			8CT	Others

Table 8. Differences in C-stretch Patterns Between 13 Pairs of Urine and Blood Samples

HV1C

HV2C

Urine

Blood

Urine

Blood

0CT>5CT

0CT

5CT>10C

5CT

5CT>10C

5CT>10C>9C

8CT

8CT>7CT

10C>11C

10C>11C>9C

10C>11C

10C>11C>9C

10C>11C

6CT>7CT

6CT

11C

Others

7CT

7CT>13C

8CT

8CT>7CT

8CT

Others

**Table 9.** Differences in C-stretch Patterns Between 5 Pairs of Urine And Blood Samples
N	HV1C	HV2C
2	10C>11C>9C	11C>10C>9C
1	10C>9C>11C	11C>10C>9C
1	10C>9C>11C	10C>11C>9C
1			7CT>6CT	6CT>7CT

Table 9. Differences in C-stretch Patterns Between 5 Pairs of Urine And Blood Samples

HV1C

HV2C

Urine

Blood

Urine

Blood

10C>11C>9C

11C>10C>9C

10C>9C>11C

11C>10C>9C

10C>9C>11C

10C>11C>9C

7CT>6CT

6CT>7CT

**Table 10.** Detection Limits of the Assay for DNA From Urine Samples Cryopreserved for 1 - 1.5 Years
Sample No.	MtDNA type	Detection limit ng as nDNA
1	A	5CT>10C	8CT	0.005
2	A	3CT	8CT	0.0025
3	B	10C>11C>9C	8CT	0.005
4	B	10C>11C>9C	7CT	0.0025
5	D4	5CT	Others	0.005
6	D4	5CT>10C	7CT	0.0025
7	D5	10C>11C>9C	7CT>6CT	0.005
8	D5	10C>11C>9C	6CT>7CT	0.0025
9	M7a	10C>11C>9C	7CT	0.005
10	Others	10C>9C>11C	7CT	0.0025

Table 10. Detection Limits of the Assay for DNA From Urine Samples Cryopreserved for 1 - 1.5 Years

Sample No.

MtDNA type

Detection limit ng as nDNA

Haplogroup

HV1C

HV2C

5CT>10C

8CT

0.005

3CT

8CT

0.0025

10C>11C>9C

8CT

0.005

10C>11C>9C

7CT

0.0025

5CT

Others

0.005

5CT>10C

7CT

0.0025

10C>11C>9C

7CT>6CT

0.005

10C>11C>9C

6CT>7CT

0.0025

M7a

10C>11C>9C

7CT

0.005

Others

10C>9C>11C

7CT

0.0025