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Amplified DNA Product Separation for Forensic Analysts

Sample Preparation

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After the samples are amplified, they are prepared for capillary electrophoresis. The samples are mixed with formamide and an internal size standard. The internal size standard is included in commercially available typing kits, and the quality of this regent is rarely a concern. Formamide can be purchased from multiple vendors and varies in quality. High-quality formamide is important for successful capillary electrophoresis.01

Read more about capillary electrophoresis in this course.

Diagram of Sample Preparation showing Hydrogen Bonds, Formamide, and Urea Heat
National Institute of Justice (NIJ) (see reuse policy).

To effectively separate the amplicons, DNA must be single-stranded. Double-stranded DNA can be modified to single-stranded DNA using chemicals, heat, or both; this process breaks the hydrogen bonds. Placing a sample in formamide will denature it. Many laboratories initially subject the sample to heat, immediately followed by snap cooling of the sample in the presence of formamide. This two-step process ensures that the DNA is denaturated.

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