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TM248 · Revised 12/18
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4.D. Data Analysis—Controls
Determine that the control reactions produced the expected results before analyzing your samples. The worksheets
provided (see Section 7.E, Table 1) can be used for analysis of both the control and experimental samples.
1.
Negative No-DNA Control:
There should be no specific amplification products in the lanes containing the
negative no-DNA control reactions. There may be some low-molecular-weight bands or smearing that are the
result of primer interactions. Amplification products in the negative no-DNA control reactions are indicative of
contaminating DNA, and the experiment should be repeated with care to avoid contamination.
2.
Positive Male Genomic DNA Control:
The number and size of the amplification products for each Multiplex
Master Mix are indicated in Table 1 (Section 7.E). The positive Male Genomic DNA control reaction for each
Multiplex Master Mix should have all the bands indicated for that Multiplex Master Mix (Table 1). The sizes of
the amplification products can be estimated by comparison with the DNA markers. If all of the expected bands
are not present in the reactions with positive Male Genomic DNA control, or if there are prominent extra bands,
it is indicative of a problem with the amplification reagents or the thermal cycler (see Section 5). Results should
not be considered valid if any of the expected amplification products are missing in the reactions with the positive
Male Genomic DNA control.
3.
Control Primer in Multiplex Master Mixes:
In Multiplexes A, B, C, and D reactions the smallest
amplification product (83bp) should be produced from an X-linked locus
(SMCX)
. In Multiplex E the largest
(496bp) amplification product should be produced from the
ZFY/ZFX
genes. The absence of these products is
indicative of a problem with that particular PCR amplification. If the control bands are present with the Male
Genomic DNA control but not with the sample DNA, it suggests that there may be a problem with the genomic
DNA used as a template, such as the presence of impurities, inaccurate DNA quantitation or degraded DNA.
Check the DNA template on an agarose gel before repeating the amplification. Repeat any reactions in which
the control product is absent. It may be necessary to isolate the template DNA again. We recommend using
the Wizard
®
Genomic DNA Purification Kit (Section 7.B; Cat.# A1120, A1125, A1620) or the MagneSil
®
KF,
Genomic System (Cat.# MD1460) for isolation of template DNA. Ideally, DNA should be eluted in water.
4.
Female DNA Control (optional):
Some researchers include a female DNA control. For Multiplexes A, B, C
and D, the smallest product (83bp) and the largest product (496bp) for Multiplex E should be present when
female DNA is used as a template. Nonspecific bands may appear when female genomic DNA is used. These
bands are generally faint and do not correspond in size to male-specific bands. With Multiplex E, a distinct high-
molecular-weight band is generally seen.
4.E. Data Analysis—Experimental Samples
The worksheets provided in the Appendix, Section 7.E, can be used for analysis of experimental samples. Determine
the presence or absence of the expected PCR products (Table 1). If there are any products absent from the reactions,
they can be mapped using the Y Chromosome Map Worksheet (Table 2). All deletions should be contiguous. Figure 4
shows an example of gel analysis of DNA carrying a Y chromosome deletion.
Adjacent regions of the Y chromosome do not appear as sequential amplification products using the Y Chromosome
Deletion Detection System. Exceptions are SY242 and SY208 of the DAZ locus, which are represented as sequential
amplification products in Multiplex B, and SY84 and SY86 of the DYS273 and DYS148 loci, which are represented as
sequential amplification products in Multiplex E.
