IBG-Hvar1

Downloadable pdf of this website Genotyping Laboratory Web site IBG Main Web site	Sections
	Primer Sequences	PCR Setup	Determination of Zygosity
	Preparation of Primer Mixture	Analysis	References
	PCR Master Mix for 20 µL reactions	IBG-Hvar1

IBG-Hvar1 is a panel consisting of 11 highly polymorphic, unlinked Short Tandem Repeat (STR or microsattelite) markers: D1S1679, D2S1384, D3S1766, D4S1627, D6S1277, D7S1808, D8S1119, D9S301, D13S796, D15S652, D20S481 and the sex determining marker, amelogenin. It is a one-tube 12-plex PCR system. This panel is used for the determination of zygosity of twin pairs, and along with IBG-Hvar2 (see), as part of a genomic control panel.

Primer Sequences and dyes for IBG-Hvar1, listed according to size of the amplified PCR product:

AMELO-F	5’- NED - CCC TGG GCT CTG TAA AGA ATA GTG –3’
AMELO-R	5'- ATC AGA GCT TAA ACT GGG AAG CTG -3'

D2S1384-F	5’- NED-AAT AGA GGG CCC TTG CTT AA -3’
D13S796-R	5'- TTT GGG ATA AAA GGT ATT TTG C -3'

D13S796-F	5' - FAM - CAT GGA TGC AGA ATT CAC AG -3'
D13S796-R	5'- TCA TCT CCC TGT TTG GTA GC -3'

D1S1679-F	5'- HEX- GCC ATC AAG AAA ACT AGT ACT GC -3
D1S1679-R	5'- ACC ATG GTA CTC AGC AGT GC -3'

D8S1119-F	5’- NED-TCA AAG CAG GTT ACT CTC ACG –3’
D8S1119-R	5'- TAA ATA TGG GAA GGC AGC AG -3'

D4S1627-F	5'- FAM- AGC ATT AGC ATT TGT CCT GG -3'
D4S1627-R	5'- GAC TAA CCT GAC TCC CCC TC -3'

D9S301-F	5'- FAM- AGT TTT CAT AAC ACA AAA GAG AAC A -3'
D9S301-R	5'- ACC TAA ATG TTC ATC AAA AGA GG -3'

D3S1766-F	5'- HEX-ACC ACA TGA GCC AAT TCT GT -3'
D3S1766-R	5'- ACC CAA TTA TGG TGT TGT TAC C -3'

D20S481-F	5’- NED-TGG GTT ATG AGT GCA CAC AG –3’
D20S481-R	5'- AAC AGC AAA AAG ACA CAC AGC -3'

D7S1808-F	5'- FAM- CAG AAC AAA CAA ATG GGG AG -3'
D7S1808-R	5'- CCA AAT AAG ACT CAG GAC GC -3'

D15S652-F	5’- NED-GCA GCA CTT GGC AAA TAC TC –3’
D15S652-R	5'- CAT CAC TCA AGG CTC AAG GT -3'

D6S1277-F	5'- HEX - ACA CTG CAG GGT AAG ACA GC -3'
D6S1277-R	5'- AAG ACA GTG TCT AAG CTG TCA CA -3'

Depending on the filter set, TET can be substituted for NED and VIC can be substituted for HEX

Preparation of Primer Mixture for PCR

Locus (color)	Size Range		Stock Concentration (µM)	µL Primer to prepare 1350 µL Pimer mix	Concentration of Primer in stock (µM)	Final Concentration using 4.4 µL per 20 µL reaction (µM)

Amelogenin	106 or 112	Forward	200	2.4	0.36	0.078
Black		Reverse	200	2.4	0.36	0.078

D2S 1384	141-161	Forward	200	18	2.67	0.587
Black		Reverse	200	18	2.67	0.587

13S 796	148-168	Forward	200	6.3	0.93	0.205
Blue		Reverse	200	6.3	0.93	0.205

D1S1679	148-168	Forward	200	18	2.67	0.587
Green		Reverse	200	18	2.67	0.587

D8S 1119	173-197	Forward	200	22.5	3.33	0.733
Black		Reverse	200	22.5	3.33	0.733

D4S 1627	177-201	Forward	200	9	1.33	0.293
Blue		Reverse	200	9	1.33	0.293

D9S 301	209-241	Forward	200	15	2.22	0.489
Blue		Reverse	200	15	2.22	0.489

D3S 1766	222-253	Forward	200	22.5	3.33	0.733
Green		Reverse	200	22.5	3.33	0.733

D20S 481	217-253	Forward	200	12	1.78	0.391
Black		Reverse	200	12	1.78	0.391

D7S 1808	252-276	Forward	200	15	2.22	0.489
Blue		Reverse	200	15	2.22	0.489

D15S 652	288-309	Forward	200	22.5	3.33	0.733
Black		Reverse	200	22.5	3.33	0.733

D6S 1277	282-306	Forward	200	18	2.67	0.587
Green		Reverse	200	18	2.67	0.587

Total volume of Primers 362.4 µL

Water or 0.01x TE 987.6 µL

Total volume of Primer Mixture
1350 µL

PCR Master Mix for 20 µL reactions
(15 µL Master mix + 5 µL DNA)

Component	1 Tube vol (µL)	100 tubes vol (µL)	Stock Concentration	Concentration in PCR Master Mix	Final Concentration in PCR
Water	4.9	490
10x buffer	2.0	200	10 x	0.133 x	1 x
MgCl2	1.4	140	25 mM	2.33 mM	1.75 mM
dNTPs	2.0	200	2.5 µM each	333 µM each	250 µM each
Primers	4.4	440	(from table)	(from table)	(from table)
AmpliTaq Gold®	0.3	30	5 Units/µL	0.02	1.5 Units

Total volume (µL)	15	1500

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PCR Setup

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Analysis

IBG-Hvar1. This panel is used routinely to assess zygosity status of twin pairs. The figure is reproduced from a run from an ABI PRISM® 3100 Genetic Analyzer. The x-axis shows size of PCR fragments in base pairs. The y-axis is relative fluorescence. The top two panels are of a pair of monozygotic twins. All loci show identical fragment sizes, and the pair is male as evidenced by double amelogenin peaks for the X (106 bp) and Y (112 bp) chromosomes. Four of the autosomal loci (D13S796, D3S1766, D20S481 and D15S652) are homozyous in both twins, and the remaining seven are heterozygous. The second two panels are of a dizygotic twin pair. Only 6 of the 11 autosomal loci have identical peak sizes (D2S1384, D1S1679, D3S1766, D20S481, D15S652 and D6S1277), and the pair is female as evidenced by the single amelogenin peak. Red peaks are size standards. It should be noted that only the size of the fragment in base pairs (the position of the peak on the x-axis) is informative, since these polymorphisms are due to the number of base repeats. The height and shape of the peaks, while often similar for a given locus, are not informative since they have to do with variables such as amount of input DNA, efficiency of amplification in each well, amount of PCR product sampled, injection efficiency, capillary-to-capillary variation, and similar issues.

Determination of Zygosity

So, what do these data mean with respect to determining if a pair of twins is monozygotic or dyzygotic? The criterion we use for assigning monozygosity is 100% concordance. If there is disagreement of even a single allele, they cannot, by definition be MZ twins. This is a very unlikely result that rarely happens. In practice if we find only two or three differences between pairs of twins, we simply repeat the whole anaysis and re-analyze. Any discrepancies are generally resolved after a single repeat. A third run of the questionable loci is also generally done, just to be sure.

Even though we use 100% concordance to assign monozygosity, how good is that really? Isn’t it still possible that the pair is not MZ afterall? To answer these questions we need to calculate the probability that a pair of DZ twins (or any siblings) will be identical at all the loci we have used. For most of us, this is not an obvious calculation; however, an excellent discussion of this topic can be found on the homepage of Dr Dale Nyholt at the Queensland Institute of Medical Research:
QIMR Genetic Epidemiology Laboratory Home > Dale's Homepage > ZygProb WWW Interface, (from which much of this discussion is excerpted), which you are encouraged to visit, and from his paper (Nyholt, 2005). His web page offers several useful links, including one to an Excel sheet (Presciuttini calculations) which can be used to calculate the probabilities that DZ twins will be identical by state (IBS=2), that is, have identical genotypes at specified loci, using an approximation method (Presciuttini et al, 2002).

The calculation requires knowledge of the expected heterozygosity (Hexp) at each locus, which can be thought of as the probability that an individual will be heterozygous (have two alleles) at that locus. High heterozygosity is indicative of a great deal of genetic variability (many alleles at the locus—this can be eight, ten or many more), while low heterozygosity indicates little genetic variability (two or three alleles) at the locus. It is expressed as a value from 0.00 (no chance of heterozygosity at the locus, e.g., there is only one allele in the population) to 1.00 (meaning there are so many alleles at that locus that for a person to be homozygous at that locus approaches impossibility).

Fortunately, we can look up the heterozygosities for many STR loci. Bear in mind that the calculated heterozygosities will differ somewhat depending on the source (and population studied), but for many STR these differences will not be great, and for our purposes, will not greatly affect the calculation for determining the probability that twins are MZ or DZ. For the eleven STR we use (the sex determininag locus, amelogenin is not used for this calculation), the heterozygosities can be found on the Invitrogen web site for mappairs: http://mp.invitrogen.com/resources/apps/mappairs/. Those values were substituted into the appropriate cells of the Excel sheet mentioned above. The data are summarized below:

Locus Name	Expected Heterozygosity (Hexp)	Probability for sharing both alleles, P(IBS=2) 0.7753 + 0.0358Hexp - 1.1771Hexp2 + .6181*Hexp3
D2S1384	0.67	0.456787420
D13S796	0.77	0.387146457
D1S1679	0.84	0.341160582
D8S1119	0.81	0.360486372
D4S1627	0.69	0.442636103
D9S301	0.75	0.400792188
D3S1766	0.86	0.328651054
D20S481	0.90	0.304663900
D7S1808	0.81	0.360486372
D15S652	0.81	0.360486372
D6S1277	0.69	0.442636103

Thus, for a twin pair, if we determine that the alleles at all 11 of these STR loci are the same, we can be more than 99.997% sure that they are MZ twins (or less than 1 chance in 45,000). And that’s better than Ivory soap.

Acknowledgement: Development of this panel was supported in part by a grant
from the National Institute of Alcohol Abuse and Alcoholism, AA014250.

References

Nyholt DR (2005) On the probability of DZ twins being concordant for two alleles. Twin Res (in preparation)

Presciuttini, S., Toni, C., Tempestini, E., Verdiani, S., Casarino, L., Spinetti, I., De Stefano, F., Domenici, R. and Bailey-Wilson, J.E. (2002) Inferring relationships between pairs of individuals from locus heterozygosities. BMC Genetics, 3: 23. View Article

To each well add	DNA	1-5 µL	(20 ng or less) Usually 1 µL DNA + 4 µL water.
	Mastermix	15 µL

	Total volume	20 µL

PCR Cycling	1x	95 C 10 min
	30x	94 C 30 sec	55 C 30 sec	72 C 60 sec
	1x	72 C 30 min
		4 C	hold

For analysis mix:	2 µL PCR product
	20 µL Hi-Di formamide
	0.5 µL Genscan 500 Rox

Samples are analyzed on an ABI PRISM® 3100 Genetic Analyzer using standard company protocols without modification

Probability of a DZ pair sharing both alleles at all markers	= 0.000022222
Percent of DZ pairs expected to share both alleles at all markers	= 0.002222203
Average certainty of twin pair being MZ (%)	= 99.9977778
Odds for MZ compared to DZ	= 45000.38548

Total volume of Primers	362.4 µL
Water or 0.01x TE	987.6 µL
Total volume of Primer Mixture	1350 µL