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Profissional Documentos
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Gold Polymerase.
References
Ewen, K.R., M. Bahlo, S.A. Treloar, D.F. Levinson, B. Mowry, J.W. Barlow, and S.J.
Foote. 2000. Identification and analysis of error types in high-throughput genotyp-
ing. Am. J. Hum. Genet. 67:727-736.
Hecker, K.H. 2003. Basics of automated, high-accuracy mutation screening with the
WAVE nucleic acid fragment analysis systems. In: Genetic Variance Detection:
Nuts & Bolts of DHPLC in Genomics (ed. K.H. Hecker), pp. 15-34. Eagleville:
DNA Press LLC.
Hoelzl, G., and P. Oefner. 2004. Microsatellite genotyping by LC/MS using the Finnigan
LTQ linear ion trap mass spectrometer. Application Note 339, Thermo Electron
Corporation.
June 27, 2013
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Holland, P.M., R.D. Abramson, R. Watson, and D.H. Gelfand. 1991. Detection of specif-
ic polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of
Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 88:7276-7280.
Huber, C.G., A. Premstaller, H. Oberacher, W. Xiao, G.K. Bonn, and P.J. Oefner. 2001.
Mutation detection by capillary denaturing high-performance liquid chromatography
using monolithic columns. J. Biochem. Biophys. Meth. 47:5-20.
Kroutil, L.C., and T.A. Kunkel. 1999 Deletion errors generated during replication of
CAG repeats. Nucleic Acids Res. 27:3481-3486.
Oberacher, H., P.J. Oefner, G. Hlzl, A. Premstaller, K. Davis, and C.G. Huber. 2002.
Re-sequencing of multiple single nucleotide polymorphisms by liquid chromato-
graphy-electrospray ionization mass spectrometry. Nucleic Acids Res. 30:e67.
Oberacher, H., H. Niedersttter, B. Casetta, and W. Parson. 2006. Some guidelines for the
analysis of genomic DNA by PCR-LC-ESI-MS. J. Am. Soc. Mass Spectrom.
17:124-129.
Oberacher, H., and W. Parson. 2007. Forensic DNA fingerprinting by liquid chromatog-
raphy-electrospray ionization mass spectrometry. BioTechniques 43:Svii-Sxiii.
Oberacher, H., F. Pitterl, G. Huber, H. Niedersttter, M. Steinlechner, and W. Parson.
2008. Increased forensic efficiency of DNA fingerprints through simultaneous reso-
lution of length and nucleotide variability by high-performance mass spectrometry.
Hum. Mutat. 29:427-432.
Wenz, H., J.M. Robertson, S. Menchen, F. Oaks, D.M. Demorest, D. Scheibler, B.B.
Rosenblum, C. Wike, D.A. Gilbert, and J.W. Efcavitch. 1998. High-precision geno-
typing by denaturing capillary electrophoresis. Genome Res. 8:69-80.
June 27, 2013
38
Xiao, W., and P.J. Oefner. 2001. Denaturing high-performance liquid chromatography: A
review. Hum. Mutat. 17:439-474.
!"#$%& (&#&)*+
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3>.-* *0)549 -4/40* ?@ABC >0/7.*+/43 (-.9 <D 4E*0)* F4G#3> 0)5 ).)1
F4G#3> /./870*#.)3 $-.8/45 #)*. <H 6783*4-3%
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/./870*#.) 83#)$ ?'C !!"#
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0)5 ?IC ).-907#J45 %&' K07843 (.- &L =1
6>-.9.3.94 9#6-.30*477#*4 7.6#%
Table 1. Samaritan and Cohen Modal Y-chromosome STR haplotypes, using typing nomenclature of
Kayser et al. (1997).
Y chromosome marker (DYS prefix)
Family 19 388 389-I 389-II 390 391 392 393 426 438 439 YCAIIa YCAIIb Haplogroup
3
Cohen-1 14 12 13 18 24 10 11 13 11 10 19 19 22 E3b (M78)
Cohen-2 14 12 13 18 24 11 11 13 11 10 19 19 22 E3b (M78)
Danfi-1 14 15 14 16 24 10 11 12 11 9 18 19 22 J2f (M172, M67)
Danfi-2 14 15 14 16 23 10 11 12 11 9 18 19 22 J2f (M172, M67)
Joshua-Marhiv-1 14 16 13 17 23 11 11 12 11 10 17 22 22 J1 (M267)
Joshua-Marhiv-2 14 16 13 17 23 11 11 12 11 10 17 22 22 J1 (M267)
Joshua-Marhiv-3 14 16 13 17 23 11 11 12 11 10 17 22 22 J1 (M267)
Joshua-Marhiv-4 14 16 13 17 23 11 11 12 11 10 17 22 22 J1 (M267)
Tsedaka-1 14 15 13 16 23 10 11 12 12 8 20 19 22 J2* (M172)
Tsedaka-2 14 15 13 16 23 10 11 12 12 8 20 19 22 J2* (M172)
Tsedaka-3 14 15 13 16 23 10 11 12 12 8 20 19 22 J2* (M172)
Tsedaka-4 14 15 13 16 23 10 11 12 12 8 20 19 22 J2* (M172)
CMH
1
14 16 13 16 23 10 11 12 11 10 19 22 22 J1 (M267)
CMH
2
14/15 15/16 14 16 23 10 11 12 11 9 19 19 22/23 J2* (M172)
1
Original Cohen Modal Haplotype (CMH) allelotypes printed in bold (Thomas et al. 1998). The allelotypes
of DYS389I&II, DYS426, DYS438, DYS439, and YCAIIa&b are the consensus observed in five
Samaritan and tweleve Cohen haplogroup J1 sequences.
2
Consensus CMH STR haplotypes associated with haplogroup J2 sequences of six Samaritans and nine
Cohanim.
3
Haplogroup assignment based on single-nucleotide polymorphisms given in parentheses (Shen et al. 2004)
Table 2. Y chromosome haplotype distances among Samaritan families.
Tribe
Levi Ephraim Manasseh
Lineage Family C1 C2 JM JM JM JM D1 D2 TS1 TS1 TS1 TS2
Cohen
C1 1 13 13 13 13 9 10 11 11 11 11
C2 12 12 12 12 10 11 12 12 12 12
Joshua-
Marhiv
JM 0 0 0 10 9 12 12 12 12
JM 0 0 10 9 12 12 12 12
JM 0 10 9 12 12 12 12
JM 10 9 12 12 12 12
Danfi
D1 1 6 6 6 6
D2 5 5 5 5
Tsedaka
TS1 0 0 0
TS1 0 0
TS1 0
TS2
Entries in the table are the total number of single-step repeat mutations between two
corresponding chromosomes. Tribes may include more than one lineage as defined by
family name. Family names are annotated as in Table 1.
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78(63$69
:6$6.);<=)5#$<>
?6&6 @#-6.5#$<>> A*4B6. )/ C++6+65
D,4,.#$,&5 EFGE0H EF0EI
JEFI0I H EFKL1M>>>
EFG0G H EFEGN KFO H EFLEL
JKF0O H EFGPPM>>>
Q#B<,& R6S5 EFLPI H EF0LI EFPLN H EFEKO 1FIK H EFGLE
T).)33,& R6S5 EFGKK H EF01P EFPGN H EFEKN 1FLL H 0FE01
U)%, EFLNLH EF0IP EFPP1 H EFE0N 1FON H EFIIE
@.*;6 EFG1N H EFEPI EFPN0 H EFENK 1FIP H EFPNL
V69)*#&5 EFIL0 H EFKOL EFP10 H EFE1E 1FG1 H 0F1GL
W.,X# R6S5 EFGIE H EFEOL 0FEEE H EFE0I NFEE H 0FEEE
7$%#)(#,& R6S5 EFG0G H EF0EP EFPLG H EFEKL 1FNI H 0F0KL
C5%Y6&,;# R6S5 EFGE0 H EF0L1 EFPLP H EFEK0 1FON H EFGLL
Z,+65$#&#,&5 EFLG1 H EF0OO EFP1O H EFE11 NF1P H 0FENN
2646&# R6S5 EFGNP H EFEOO EFPPO H EFE0G 1FON H EFGLL
* Heterozygosity is corrected to be comparable to autosomal values using the formula
H
corr
= 4H
uncorr
/(3H
uncorr
+1) for each locus; means and standard deviations are taken
across corrected locus values.
** Sample-size corrected value standard deviation
*** Average over 13 markers including three monomorphic markers
Table 4. Within-population variation for 15 autosomal microsatellites*
Expected
Heterozygosity
Number of Alleles
Samaritans 0.616 0.174 4.067 1.552
Libyan Jews 0.702 0.075 5.267 2.738
Moroccan Jews 0.738 0.063 5.667 1.320
Cohanim 0.714 0.056 5.333 1.792
Druze 0.714 0.079 5.333 2.469
Bedouins 0.726 0.059 6.200 2.631
Iraqi Jews 0.719 0.065 4.933 2.336
Ethiopian Jews 0.763 0.063 5.867 2.446
Ashkenazi Jews 0.724 0.088 5.467 1.821
Palestinians 0.730 0.054 6.333 2.789
Yemeni Jews 0.697 0.092 5.333 2.658
* Allowable level of missing data was set to 0.09 to allow 15 rather than 13 loci to be
included for calculations; estimates standard deviation; sample-size corrected values
standard deviation
Table 5. Genetic distances of Samaritans from other populations
F
ST
Neis D
(D corrected for sample size)
(!!)
2
Y* Y
Haplotypes*
Autosomes Autosomal
SNPSTRs
Y Autosomes Autosomes
SNPSTRs
Y Autosomes**
Libyan
Jews
0.050 0.027 0.047 0.047
0.227
(0.160)
0.065
(0.01)
0.072
(0.011)
0.292 0.510
Moroccan
Jews
0.038 0.025 0.045 0.039
0.172
(0.102)
0.056
(-0.003)
0.049
(-0.016)
0.422 0.493
Cohanim 0.021 0.024 0.054 0.057
0.072
(0.021)
0.078
(0.029)
0.096
(0.041)
0.076 0.378
Druze 0.055 0.032 0.056 0.050
0.260
(0.185)
0.081
(0.022)
0.078
(0.012)
0.651 0.441
Bedouin 0.041 0.033 0.036 0.045
0.128
(0.083)
0.049
(0.006)
0.072
(0.025)
0.208 0.366
Iraqi Jews 0.031 0.023 0.054 0.050
0.136
(0.059)
0.079
(0.025)
0.079
(0.020)
0.397 0.540
Ethiopian
Jews
0.072 0.027 0.061 0.067
0.349
(0.275)
0.076
(0.0)
0.103
(0.018)
0.957 0.906
Askenazi
Jews
0.034 0.026 0.058 0.052
0.143
(0.076)
0.086
(0.037)
0.084
(0.031)
0.425 0.507
Palestinian 0.074 0.032 0.043 0.041
0.355
(0.308)
0.057
(0.014)
0.059
(0.011)
0.599 0.414
Yemeni
Jews
0.025 0.024 0.072 0.069 0.106
(0.033)
0.114
(0.063)
0.120
(0.064)
0.315 0.517
* Y F
ST
values are corrected to be comparable to autosomal values using the formula F
STcorr
= F
STuncorr
/(4-3F
STuncorr
).
Wilcoxon signed-rank test comparing F
ST
values across populations (excluding Ethiopians) for autosomes vs. Y (based on separate microsatellite loci): p-value =
0.25.
Table 6. F
ST
genetic distances per locus for the indicated population comparisons
a
.
Samaritans vs. Jews* Samaritans vs. Non-Jews
Y chromosome
marker
F
ST
uncorrected F
ST
corrected** F
ST
uncorrected F
ST
corrected**
DYS19 0.234 0.071 0.281 0.089
DYS388 0.174 0.050 0.327 0.108
DYS389I 0.036 0.009 0.143 0.040
DYS389II 0.002 0.001 0.072 0.019
DYS390 0.080 0.021 0.154 0.044
DYS391 0.086 0.023 -0.021 -0.005
DYS392 0.091 0.024 0.131 0.0363
DYS393 0.100 0.027 0.300 0.097
DYS426 0.043 0.011 0.189 0.055
DYS438 0.081 0.022 0.212 0.063
DYS439 0.117 0.032 0.210 0.0621
YCAII/1 0.051 0.013 0.071 0.019
YCAII/2 0.206 0.061 0.302 0.098
Mean 0.028 0.056
Autosomal
Marker
F13B 0.183 0.146
TPOX 0.148 0.149
D2S1400 0.079 0.077
D3S1358 0.031 0.031
D4S2361 0.030 0.025
D5S1456 0.031 0.024
5SR1 0.051 0.029
D7S2846 0.014 -0.0005
D8S1179 0.025 0.009
D10S1426 0.016 0.020
GATA48 0.141 0.150
D13S317 0.113 0.052
FES 0.025 0.010
D16S539 0.004 0.050
D17S1298 -0.010 -0.017
Mean 0.059 0.050
*Ethiopian Jews were excluded for this analysis.
**F
ST
values for Y chromosomes corrected for comparison to autosomes as in Table 5. Two-sided
Wilcoxon signed-rank tests (setting negative values to 0): Autosomes p = 0.103, Y chromosome p = 0.003.
a. All F
ST
distances from Arlequin (3.5).
Table 7. Genetic distances of Samaritans from other populations, grouped into Jewish
a
and Non-Jewish subsets.
F
ST
Neis D
(D corrected for sample size)
Y* Y haplotypes* Autosomes** Autosomes
SNPSTRs
Y Autosomes Autosomes
SNPSTRs
Samaritans vs.
Jewish
0.023 0.021 0.044 0.040
0.112
(0.073)
0.069
(0.04)
0.069
(0.036)
Samaritans vs.
Non-Jewish
0.041 0.025 0.039 0.037
0.198
(0.158)
0.056
(0.025)
0.058
(0.022)
Jewish vs.
Non-Jewish
0.009 0.003 0.003 0.003
0.048
(0.035)
0.006
(-0.005)
0.007
(-0.005)
a. Ethiopian Jews are excluded from this analysis.
* Y F
ST
values are corrected to be comparable to autosomal values as in Table 5.
Table 8. Assignment of Affinity Propagation based clusters derived from 13 Y-chromosomal short tandem repeat loci and depicted in
Figure 1 to their respective Jewish and non-Jewish populations Y-SNP based haplogroups.
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F8&G( D?E9 ;-5< / - , . . , , , , .
H"8"II&) D?E9 ;-5< - - / - - / . ,
J(KL&) D?E9 ;-5< , , 1 . , - / -
M?7?)( D?E9 ;-5< , 0 . 0 , , . ,
N'=("#(&) D?E9 ;,2< 2 , , . 0
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!&'=&) ;-5< - , . 4 , , , -
Q&%&9= ;-5< , 0 - / / /
>8&=$( ;-/< , , , . / 3 . , , ,
>$8$9=" ;-5< - - ,, 0
F'&%(&)9 ;-4< ,5 . ,5 , 0
R$99(&)9 ;-.< / 3 - . 1
M"8$K& ;-/< / - 0 3 . , ,
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F!($ V!"$ W!)$ S!($ >!($ >!"$ R!"$ V!"$ V!"$ V!'$ F!($ *!($ *!"$ N!($ F!($ U!"$ R!"$ ON!'$
R!"$ J!)$ F!)$ U!"$ W!)$ >!"$ ON!"$ D!"$ ON!"$ S!"$ U!($
S!"$ *!"$ X!"$ D!"$ X!"$ F!"$ >!"$
F!"$ N!"$ D!"$ *!"$
Q!"$ X!"$
!"##$%&%'()$ +),$% -. Cen8ank accesslon numbers, nucleoLlde poslLlons of Lhe 3' ends of Lhe forward prlmers ln Cen8ank accesslons, ranges of observed allele slzes and correspondlng
numbers of repeaLs, and sequences of Lhe forward and reverse prlmers employed for Lhe ampllflcaLlon of Lhe 13 ?-chromosome and 14 auLosomal S18 locl sLudled.
S18
Cen8ank
Accesslon no.
oslLlon
Allele slzes
observed (bp)
no. of
repeaLs
lorward prlmer (3'-3') 8everse prlmer (3'-3')
u?S19 Al 140632 1 131-173 11-17 C1AC1CAC11C1C11A1AC1C11111 A1C1CCC11AACCACAC1C1CAC
u?S388 AC 004810 62380 130-174 10-18 CAA11CA1C1CAC11ACCCC111ACC
1
CACCCCCACC1111AC1CAC
1
u?S389-l Al 140633 1 146-166 10-13 CCAAC1C1CA1C1C1A11A1C1A1C
1
C1AACAACACCA1CAC1CCC1A11C
1
u?S389-ll 270-290 13-21
u?S390 Al 140636 19 132-168 17-26 CCCC1CCA1111CC1AC CACAAACAACCAAACA1ACA1ACA1C
u?S391 nC 002806.1 24917 136-132 8-12 C1A1CA1CCA1CC11A1C1C11C1 A11CCCA1ACACCCA1ACC1ACC
u?S392 Al 140638.1 23 133-131 9-16 CAAC1AA111CA111CAAC1C111C ACC1ACCAA1CCCA11CC11AC
u?S393 Al140639 1 113-131 11-13 C1CC1C11C1AC11C1C1CAA1AC
1
AAC1CAAC1CCAAAAAA1CACC
1
u?S426 AC 007034 133374 88-97 10-13 C1CAAAC1A1CAAACCA1CACCA
1
C1C111CACACCACAACAC1CC
1
u?S438 AC 002331 129799 211-236 8-13 1CCCCAA1AC11CAACCC1AA C1CCCACACCCC1A1AA1CC
u?S439 AC 002992 91172 203-223 16-21 1CCAC11C11A1CC1111ACC1C1
1
CCCA1111C11AACC11CCC1C
1
?CAll a+b AC013978 79863 144-138 16-23 1C1CAAAA111AACCCACAA1CA
1
CCA11CCAA1ACCAC111C1CACC
1
l138 AAuC01009326.1 36818 169-183 6-10 1CACC1CC1C1AC1ACCA1A
2
CA1CA1CCCA11CCAC1C1AC
2
1Cx M68631 1817 114-130 8-12 CAC1ACCACCCACAACCC1CC
2
CC1CCCAACACCCACCA1CAC
2
u2S1400 A?083997 338 111-139 7-14 1CCAA1CC1111ACC1C1CCC1CC
3
CA1ACC1CAACCA1AAC1CA11CC
3
u3S1338 AC099339.2 77721 119-143 13-19 AC1CCAC1CCAA1C1CCC1
2
A1CAAA1CAACACACCC11C
2
u4S2361 AC079160.3 38789 136-162 7-16 CCACC1CAC111CA11ACCC
3
ACACCA1CA1CCCCCA1C
3
3S81 AC026743.4 147644 136-174 13-22 C11AAA1ACAC1C1CC1AC111C
3
A1CC1A1CA11AC1ACC1AAC1ACC
3
u3S1436 AC008680.3 172273 182-218 6-13 1A1CCAA11C1AACCCCC11
3
CC1CCAAAACCC1AA11C1CC
3
u7S2846 AC073068 93318 170-190 10-13 1C1AAAC1CC111CCACAC1C
3
ACA1C1C1CCA1CAAA1CA1C
3
u8S1179 AC100838.3 140061 161-201 8-18 11111C1A111CA1C1C1ACA11CC
2
CC1ACC1A1AA11AC11CA1111CA
2
u10S1426 AL360172 131699 146-174 8-13 11CC1CC1C1CA1CC1C111
3
C1C11AAC1CA111CCCCCA
3
CA1A48L08 AC087783 93228 113-143 7-14 CA1CCA1C1CA1CCCA1CA11
4
11CACCC1AC1CCCAAC11C
4
u13S317 AL391334.12 16762 173-197 9-13 ACACAAC1C1CCCA1C1CCA
2
CCCCAAAAACACACACACAA
2
lLS/lS AC124248 131132 142-166 8-14 CCAACA1CCAC1CCC1C11A
2
C1CCACCC1CCCCAAACAA1
2
u16S339 C07293 224 141-169 7-14 CA1CCCAACC1C11CC1C11
2
ACC111C1C1C1CCA1C1C1
2
u17S1298 AAuC01128113 48874 128-144 7-11 CCACCC1AC1AAC1ACCA1CC C111CAC1CCC1ACCA1CC
rlmer sequences were obLalned from
1
8uLler eL al. (2002),
2
hLLp://www.csLl.nlsL.gov/bloLech/sLrbase/seq-lnfo.hLm,
3
hLLp://www.ncbl.nlm.nlh.gov, and
4
hLLp://www.genome.ucsc.edu.
!"##$%&%'()$ +),$% -. LxpecLed molecular masses of repeaL moLlfs and of Lhe shorLesL alleles observed for forward and reverse sLrands,
respecLlvely.
!+. &/(01
1/2
0'32%&%'( 4567 89
1/2
45)7
:
&/(01
2%;
0'32%&%'( 45)7 89
2%;
45)7
:
u?S19 1ACA 1239.82 46843.30 A1C1 1210.79 46296.22
u?S388 A11 921.60 46234.93 1AA 930.62 46319.08
u?S389-l [1C1C] [1C1A] [1226.78][1210.78] 44316.34 [ACAC] [ACA1] [1244.78][1239.82] 43731.80
u?S389-ll [1C1C] [1C1A] [1226.78][1210.78] 80873.33 [ACAC] [ACA1] [1244.78][1239.82] 83344.31
u?S390 [1C1C] [1C1A] [1226.78][1210.78] 41347.72 [ACAC] [ACA1] [1244.78][1239.82] 41284.97
u?S391 1C1A 1210.78 41180.67 ACA1 1239.82 42713.82
u?S392 1A1 921.60 40136.16 A1A 930.62 40011.19
u?S393 ACA1 1239.82 33663.19 1C1A 1210.78 33247.99
u?S426 C11 937.60 28037.17 CAA 913.60 28037.31
u?S438 1111C 1303.97 64736.81 AAAAC 1382.04 63301.36
u?S439 CA1A 1239.84 63989.71 C1A1 1210.79 62314.60
?CA ll CA 602.40 44213.93 C1 633.40 44609.90
l138 AAA1 1243.83 32230.92 A111 1223.80 32040.89
1Cx AA1C 1239.82 33334.99 CA11 1210.78 34976.60
u2S1400 [CC11][CC1C] [1186.76][1211.77] 33612.63 [CCAA][CCAC] [1284.83][1260.81] 34832.63
u3S1338 ACA1 1239.82 36881.99 A1C1 1210.78 36311.68
u4S2361 1A1 921.60 42370.62 A1A 930.62 42334.73
3S81 CA 602.40 47839.13 C1 633.40 48407.41
u3S1436 CA1A 1239.82 61291.91 1A1C 1210.78 60886.62
u7S2846 C1A1 1210.78 31863.74 A1AC 1239.82 33017.38
u8S1179 [1C1A][1C1C] [1210.78][1226.78] 49393.24 [1ACA][CACA] [1239.82][1244.78] 49733.37
u10S1426 CA1A 1239.82 43270.33 1A1C 1210.78 44796.03
CA1A48L08 CA1A 1239.82 33062.73 1A1C 1210.78 33836.36
u13S317 CA1A 1239.82 33722.96 1A1C 1210.78 33030.32
lLS/lS A111 1223.80 44281.70 AAA1 1243.83 43318.21
u16S339 CA1A 1239.82 43629.42 1A1C 1210.78 43333.03
u17S1298 [AA1C][AACC] [1239.82][1204.79] 39303.38 [11CA][CC11] [1210.78][1266.80] 39646.70
:
1heoreLlcal molecular mass of smallesL fragmenL observed wlLh Lhe leasL number of repeaLs based on reference sequence found ln Cen8ank.
!"##$%&%'()$ +),$% -. naLure and genomlc locaLlon of slngle-base subsLlLuLlons
observed wlLhln or ad[acenL Lo shorL Landem repeaLs u?S438 and u?S393.
u?S438 (Cenbank Accesslon no. AC002331) oslLlon Marker lu*
8ef. -cr-1111C1111 C [1111C]
6
-cr- A -cr- C -cr-
1 -cr-1111C1111 / [1111C]
6
-cr- A -cr- C -cr- g.129837
2 -cr-1111C1111 C [1111C]
6
-cr- 0 -cr- C -cr- g.129884 M393
3 -cr-1111C1111 C [1111C]
6
-cr- A -cr- / -cr- g.129884 M391
u?S393 (Cenbank Accesslon no. Al140639) oslLlon Marker lu*
8ef. gLggLcLLcLacLLgLgLcaaLac A CA1 (ACA1)
14
-cr-
1 gLggLcLLcLacLLgLgLcaaLac 0 CA1 (ACA1)
11
-cr- g.26 M380
*SLanford numberlng sysLem
Supplemental Figure 1. Impact of different Taq polymerases on the spectral quality of a
184-bp amplicon: (a) Optimase
TM
, Transgenomic, Omaha, NE; (b) Discoverase
TM
dHPLC
DNA Polymerase, Life Technologies, Invitrogen, Carlsbad, CA; (c) AmpliTaq
Gold, Life
Technologies. The upper row shows the reconstructed ion chromatograms: within the first 1.5
minutes unincorporated deoxynucleotides and primers elute from the column, followed by a
peak at about 3.5 minutes, which contains the two chromatographically not resolved single-
stranded components of the PCR amplicon of interest. The lower row shows the deconvoluted
mass spectra of the amplicon. The two major signals in the deconvoluted mass spectra
represent the mass spectrometrically resolved forward and reverse strands of the amplified
DNA and their respective molecular masses in Dalton. The differences in signal-to-noise ratio
reflect differences in proofreading capability, absence of 3'-adenylation activity, and
polymerase storage buffer composition.
Enzyme
Mw
for
[Da]
(theor.)
Mw
rev
[Da]
(theor.)
Mw
for
[Da]
(meas.)
Mw
rev
[Da]
(meas.)
Dev
for
[Da]
Dev
rev
[Da]
Optimase
mutant
69254.71 70247.70
69236 70265
-18.71
(-270 ppm)
17.30
(246 ppm)
wild type 69249 70248
-5.71
(-82 ppm)
0.30 (4
ppm)
AmpliTaq
Gold
mutant
69567.92 70560.91
69565 70557
-2.92
(-4 ppm)
-3.91
(-5 ppm)
wild type 69573 70565
5.08 (7
ppm)
4.09 (6
ppm)
Supplemental Figure 2. Sequence traces confirm the presence of an A>G transversion
next to the 3! terminus of the primer detected after amplification with Optimase, a
proofreading enzyme with 3!-5! exonuclease activity (a, b, d), while the single nucleotide
polymorphism went undetected after amplification with AmpliTaq Gold, that lacks 3!-5!
exonuclease activity (c). (a) Optimase
TM
, mutant, short product, (b) Optimase
TM
, mutant,
long product, (c) AmpliTaq