Slot Blot Quantitation

Probably the most commonly used method in forensic labs today for genomic DNA quantitation is the so-called 'slot blot' procedure. This test is specific for human and other primate DNA due to a 40 base pair (bp) probe that is complementary to a primate-specific alpha satellite DNA sequence D17Z1 located on chromosome 17 (Waye et al. 1989, Walsh et al. 1992). The slot blot assay was first described with radioactive probes (Waye et al. 1989) but has since been modified and commercialized with chemiluminescent or colorimetric detection formats (Walsh et al. 1992).

Slot blots involve the capture of genomic DNA on a nylon membrane followed by addition of a human-specific probe. Chemiluminescent or colorimetric signal intensities are compared between a set of standards and the samples (Figure 3.3). Slot blot quantitation is a relative measurement involving the comparison of unknown samples to a set of standards that are prepared usually via a serial dilution from a DNA sample of known concentration. While comparison of the

Figure 3.3

Illustration of a human DNA quantitation result with the slot blot procedure. A serial dilution of a human DNA standard is run on either side of the slot blot membrane for comparison purposes. The quantity of each of the unknown samples is estimated by visual comparison to the calibration standards. For example, the sample indicated by the arrow is closest in appearance to the 2.5 ng standard.

Calibration standards

,-A-N

Calibration standards

20 ng

m

-2.5 ng *

-

0.63 ng

10 ng

1.25 ng

5 ng

2.5 ng

2.5 ng

5 ng

1.25 ng

-

10 ng

0.63 ng

-

20 ng

DNA standards and unknown samples on the slot blot membrane is often performed visually and therefore influenced by subjectivity of the analyst, digital capture and quantification of slot blot images has been demonstrated with a charged-coupled device (CCD) camera imaging system (Budowle et al. 2001).

Typically about 30 samples are tested on a slot blot membrane with 6-8 standard samples run on each side of the membrane for comparison purposes. For example, the standards might be a serial dilution of human DNA starting with 20 ng, 10 ng, 5ng, 2.5 ng, etc. Typically only 5 ||L of DNA extract is consumed for this quantification test.

The assay takes several hours to perform but is fairly sensitive as it can detect both single-stranded and double-stranded DNA down to levels of approximately 150 pg or about 50 copies of human genomic DNA. A 150pg DNA standard can be detected after only a 15 minutes exposure to X-ray film (Walsh et al. 1992). With chemiluminescent detection, the sensitivity can be extended below 150 pg by performing longer exposures to the X-ray film and blotting additional low dilution DNA standards on the membrane. Levels of 10-20 pg have been reported with a three-hour exposure (Walsh et al. 1992). Even when no results are seen with this hybridization assay, some forensic scientists still go forward with DNA testing and often obtain a successful STR profile. Thus, the slot blot assay is not as sensitive as would be preferred.

As of early 2004, the slot blot assay is available as a commercial product from Applied Biosystems (Foster City, CA) called the QuantiBlot Human DNA Quantitation Kit. This kit uses a DNA probe from chromosome 17 and is thus useful for determining the level of human DNA present in a sample. It is important to note though that this assay, as with most 'human-specific' tests, works on primates such as chimpanzees and gorillas due to similarities in human and other primate DNA sequences.

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  • Leilani
    What is slot blot dna quantitation?
    1 year ago

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