Slot blot hybridization was the most commonly used method until recently. The majority of laboratories used the commercially available QuantiBlot^® kit, which employs the following procedures:

• Extracted DNA is denatured and the single-stranded DNA is bound to a positively charged nylon membrane.
• After the DNA is bound to the membrane, a probe complementary to the D17Z1 locus (present in high quantities in higher primates) is applied and allowed to hybridize to the DNA.
• The hybridized complex is detected by one of several methods.⁰⁴
• The amount of DNA in the sample is estimated by comparison of the density of the band or bands observed to that of the standards.

Detection methods include:

• Colorimetry, usually employing tetramethylbenzidine (TMB), which yields a blue color when oxidized by hydrogen peroxide.
• Chemiluminescence, for example ACES™ (Gibco BRL) and™ (Amersham Biosciences). The chemiluminescent reactions cause the release of photons that are captured on film or a digital imaging device. Chemiluminescence is more sensitive than colorimetry and can detect down to 10 to 20 pg of DNA.⁰⁵

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