IBG-Hvar2

Primer Sequences and dyes for IBG-Hvar2, listed according to size of the amplified PCR product:

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

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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
Blue		Reverse	200	2.4	0.36	0.078
D3S1358	110-142	Forward	200	6	0.89	0.196
Green		Reverse	200	6	0.89	0.196
D5S818	115-150	Forward	200	8	1.19	0.262
Black		Reverse	200	8	1.19	0.262
VWA	126-166	Forward	200	5	0.74	0.163
Blue		Reverse	200	5	0.74	0.163
D4S2639	162-182	Forward	200	7.5	1.11	0.244
Green		Reverse	200	7.5	1.11	0.244
D13S317	170-204	Forward	200	9	1.33	0.293
Black		Reverse	200	9	1.33	0.293
D9S934	206-230	Forward	200	7.5	1.11	0.244
Green		Reverse	200	7.5	1.11	0.244
D8S1179	200-240	Forward	200	15	2.22	0.489
Blue		Reverse	200	15	2.22	0.489
D7S820	215-245	Forward	200	18	2.67	0.587
Black		Reverse	200	18	2.67	0.587
TPOX	260-290	Forward	200	12	1.78	0.392
Blue		Reverse	200	12	1.78	0.392
D16S539	261-305	Forward	200	12	1.78	0.391
Black		Reverse	200	12	1.78	0.391
D20S470	264-314	Forward	200	7.5	1.11	0.244
Green		Reverse	200	7.5	1.11	0.244
CSF1PO	313-353	Forward	200	8	1.19	0.262
Black		Reverse	200	8	1.19	0.262
D15S657	320-360	Forward	200	7.5	1.11	0.244
Blue		Reverse	200	7.5	1.11	0.244

Total volume of Primers	280.8 µL
Water or 0.01x TE	1069.2 µL
Total volume of Primer Mixture	1350 µL

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

PCR Setup

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 11 min
	1x	96 C 1 min
	10x	94 C 30 sec	60 C 30 sec	70 C 45 sec
	20x	90 C 30 sec	60 C 30 sec	70 C 45 sec
	1x	60 C 30 min
		4 C	hold

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IBG-Hvar2

IBG-Hvar2. This panel is used as part of a genomic control panel along with IBG-Hvar1. It is basically a home-made CODIS (Combined DNA Index System) panel consisting of the 13 markers plus amelogenin that are used for forensic DNA identification. 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 panel is the result from a genomic DNA sample from a male as evidenced by the double amelogenin peaks for the X (106 bp) and Y (112 bp) chromosomes. The bottom panel is the result from a genomic DNA sample from a female as evidenced by the single 106 bp amelogenin peak for the X chromosome. Red peaks are size standards (100, 139, 150, 160, 200, 250, 300, 340 and 350 bp). 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.

Modifications to the Panel.

As described above, IBG-Hvar2 was designed using the forensic CODIS panel as a starting point. The primer sequences used in the commercial Promega PowerPlex®16 system were published (Krenke et al, 2002) and it was simply a matter of combining them in proportions that yielded 14 analyzable amplicons. Four of the CODIS loci, THO1, D21S11, D18S51 and FGA were replaced in our panel. These four loci, normally tetranucleotide repeats, have large numbers of irregular one, two and three base pair alleles. Although these are very useful for individual identification, they are more difficult to analyze. We replaced these with four other tetranucleotide repeat STRs, D4S2639, D9S934, D20S470 and D15S657. These four were chosen because they had high heterozygosities, produced the same sized amplicon as the marker they replaced, had similar primer melting temperatures and for lack of interactions among the retained primers.

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Determination of Zygosity

We do not routinely use this (CODIS) panel to determine zygosity status, although we could. We use IBG-Hvar1 for this purpose, and have done so since 1995. The reason we do not use it is that one-tube versions of the assay were only available from commercial suppliers, and the cost was simply too great for us to use routinely. The commercial kits (ABI AmpFlSTR® Profiler Plus® and Promga PowerPlex®16 system, among others) are used for forensic applications, and one pays for the added controls and expertise they provide (and rightly so if the information is to be used in court). IBG-Hvar2 was developed in house in January of 2005 as a cost-effective method to be used along with IBG-Hvar1 in a genomic control panel.

The rationale and methods for calculating the probability that two twins are not monozygotic are given in the text for IBG-Hvar1 (see), and will not be repeated here. Expected heterozygosities for the 13 loci (amelogenin is not used for this calculation) were taken from Butler et al (2003). Using the Excel workbook developed by Dr. Dale Nyolt (QIMR Genetic Epidemiology Laboratory Home > Dale's Homepage > ZygProb WWW Interface), to calculate the probabilities that DZ twins will be identical by state (IBS=2; Presciuttini et al, 2002), we find:

These data allow the following conclusions:

Thus, for a twin pair, if we determine that the alleles at all 13 of these STR loci are the same, we can be more than 99.999% sure that they are MZ twins (or less than 1 chance in 82,000). And that is still better than Ivory soap.

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Determination of Individual Identity

This panel is used routinely in forensic laboratories to match evidentiary DNA samples to individuals. How good is it for this purpose? Very good indeed. Using the approximation method of Presciuttini et al (2002) to determine the probability that two unrelated individuals will be indentical by state for both alleles at all 13 CODIS markers, we find:

These data allow the following conclusions:

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References

Butler, J.M., Scholske, R., Vallone, P.M., Redman, J.W. and Kline, M.C. (2003). Allele Frequencies for 15 Autosomal STR Loci on U.S. Caucasian, African American, and Hispanic Populations. Journal of Forensic Science, 48: 908-911.

Krenke, B.E., Tereba, A., Anderson, S.J., Buel, E., Culhane, S., Finis, C.J., Tomsey, M.S., Zachetti, J.M., Masibay, A., Rabbach, D.R., Amiott, E.A. and Sprecher, C.J. (2002). Validation of a 16-Locus Fluorescent Multiplex System. Journal of Forensic Science, 47: 1-13, 2002,

Mizutani, M., Yamamoto, T., Torii, K., Kawase, H., Yoshimoto, T., Uchihi, R.,Tanaka, M., Tamaki, K.,  and Katsumata, Y. (2001) Analysis of 168 short tandem repeat loci in the Japanese population, using a screening set for human genetic mapping. Journal of Human Genetics, 46: 448-455.

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 bewteen pairs of individuals from locus heterozygosities. BMC Genetics, 3: 23.

Ruitberg, C.M., Reeder, D.J., Butler, J.M. (2001). STRBase: a short tandem repeat DNA database for the human identity testing community. .Nucleic Acids Res. 29: 320-322

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