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

Tables

Table 1. PCR Primer Sequences
 
TargetForwardReverseAmplicon size (bp)
PositionSequence (5′ to 3′)PositionSequence (5′ to 3′)
SNP and deletion [30]
  3010G>A2698AGAGGCGGGCATAACACAGCA3066GATCACGTAGGACTTTAATCGTTGA369
  4386T>C4344TCGAACCCATCCCTGAGAATCC4577GTTTATTTCTAGGCCTACTCAGGTAA234
  5178C>A4989CAGCTACGCAAAATCTTAGCATAC5257TTGGGCAAAAAGCCGGTTAGCG269
  8272C>del8153GGGGTATACTACGGTCAATGCTC8530TCATTTTGGTTCTCAGGGTTTGTTAT378
  8794C>T8628CAAATATCTCATCAACAACCGACTA8994CAGGGCTATTGGTTGAATGAGTAG367
  10397A>G10277ACCCCTACCATGAGCCCTACAA10515GTGAGATGGTAAATGCTAGTATAATAT239
C-stretch [36]
  HV1C15896CAAATGGGCCTGTCCTTGTA16414TGTGCGGGATATTGATTTC519
  HV2C29GGTCTATCACCCTATTAACCAC408CTGTTAAAAGTGCATACCGCCA380
Others
  ND1 [38]3344TTCTAATCGCAATGGCATTCCT3452AAGGGTTGTAGTAGCCCGTAG109
  D17Z1 [37]126TTTTGCAGGATCTACAAGTGGA332AAGAGGTCTACATGTCCCCTTG207

 

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
 
GroupSequence (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 4. ASPE Primer Sequences for Detection of HV2C Groups in MtDNA
 
GroupSequence (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
 
HV1CHV2CABD4D5M7aOthersFrequency N (%)
0CT8CT11 (0.7)
3CT7CT213 (2.0)
8CT44 (2.7)
8CT>9CT11 (0.7)
5CT7CT11 (0.7)
8CT22 (1.3)
Others11 (0.7)
10C7CT11 (0.7)
11C8CT>7CT11 (0.7)
13C>11C11 (0.7)
3CT>4CT9CT>8CT11 (0.7)
5CT>8CT7CT11 (0.7)
5CT>10C7CT204630 (20.0)
8CT21521029 (19.3)
7CT>8CT11 (0.7)
8CT>7CT617 (4.7)
8CT>9CT22 (1.3)
9CT>8CT112 (1.3)
Others112 (1.3)
10C>5CT8CT22 (1.3)
8CT>7CT11 (0.7)
10C>11C6CT11 (0.7)
8CT1113 (2.0)
Others224 (2.7)
5CT>10C>9C7CT1113 (2.0)
8CT>7CT123 (2.0)
8CT>9CT11 (0.7)
10C>3CT>9C8CT11 (0.7)
10C>11C>9C7CT121913 (8.7)
8CT521412 (8.0)
6CT>7CT11 (0.7)
7CT>13C11 (0.7)
8CT>7CT11 (0.7)
Others325 (3.3)
11C>10C>9C7CT11 (0.7)
8CT11 (0.7)
13C>9CT11 (0.7)
Others11 (0.7)
Others7CT22 (1.3)
8CT11 (0.7)
N11165651448150
%7.310.737.33.39.332.0

 

Table 6. Frequency of HV1C Pattern in the Major Haplogroups for Japanese Populations
 
HV1CABD4D5M7aOthersFrequency N (%)
0CT11 (0.7)
3CT718 (5.3)
5CT314 (2.7)
10C11 (0.7)
11C112 (1.3)
3CT>4CT11 (0.7)
5CT>8CT11 (0.7)
5CT>10C343101773 (48.7)
10C>5CT123 (2.0)
10C>11C3238 (5.3)
5CT>10C>9C3317 (4.7)
10C>3CT>9C11 (0.7)
10C>11C>9C104211633 (22.0)
11C>10C>9C224 (2.7)
Others123 (2.0)

 

Table 7. Frequency of HV2C Pattern in the Major Haplogroups for Japanese Populations
 
HV2CABD4D5M7aOthersFrequency N (%)
6CT11 (0.7)
7CT222562055 (36.7)
8CT7719321856 (37.3)
6CT>7CT11 (0.7)
7CT>8CT11 (0.7)
8CT>7CT83213 (8.7)
7CT>13C11 (0.7)
8CT>9CT1124 (2.7)
9CT>8CT123 (2.0)
13C>9CT11 (0.7)
13C>11C11 (0.7)
Others521513 (8.7)

 

Table 8. Differences in C-stretch Patterns Between 13 Pairs of Urine and Blood Samples
 
NHV1CHV2C
UrineBloodUrineBlood
10CT>5CT0CT
25CT>10C5CT
15CT>10C5CT>10C>9C8CT8CT>7CT
110C>11C10C>11C>9C
110C>11C>9C10C>11C
110C>11C>9C10C>11C6CT>7CT6CT
111COthers
17CT7CT>13C
28CT8CT>7CT
18CT>7CT8CT
18CTOthers

 

Table 9. Differences in C-stretch Patterns Between 5 Pairs of Urine And Blood Samples
 
NHV1CHV2C
UrineBloodUrineBlood
210C>11C>9C11C>10C>9C
110C>9C>11C11C>10C>9C
110C>9C>11C10C>11C>9C
17CT>6CT6CT>7CT

 

Table 10. Detection Limits of the Assay for DNA From Urine Samples Cryopreserved for 1 - 1.5 Years
 
Sample No.MtDNA typeDetection limit ng as nDNA
HaplogroupHV1CHV2C
1A5CT>10C8CT0.005
2A3CT8CT0.0025
3B10C>11C>9C8CT0.005
4B10C>11C>9C7CT0.0025
5D45CTOthers0.005
6D45CT>10C7CT0.0025
7D510C>11C>9C7CT>6CT0.005
8D510C>11C>9C6CT>7CT0.0025
9M7a10C>11C>9C7CT0.005
10Others10C>9C>11C7CT0.0025