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DNA Extraction and Quantitation for Forensic Analysts

Slot Blot Hybridization

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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.
Illustration of Slot Blot Hybridization
National Institute of Justice (NIJ) (see reuse policy).

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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