Detection Of Saliva Stains

A presumptive test for amylase is used for indicating the presence of saliva, which is especially difficult to see since saliva stains are nearly invisible to the naked eye. Two common methods for estimating amylase levels in forensic samples include the Phadebas test and the starch iodine radial diffusion test (Shaler 2002). Saliva stains may be found on bite-marks, cigarette butts, and drinking vessels (Abaz et al. 2002, Shaler 2002). A molecular biology approach using messenger RNA profiling is also being taken to develop sensitive and specific tests for various body fluids including saliva (Juusola and Ballantyne 2003). Such a molecular biology test should be able to assay blood, semen, and saliva simultaneously with great specificity and sensitivity.

DNA EXTRACTION

A biological sample obtained from a crime scene in the form of a blood or semen stain or a liquid blood sample from a suspect or a paternity case contains a number of substances besides DNA. DNA molecules must be separated from other cellular material before they can be examined. Cellular proteins that package and protect DNA in the environment of the cell can inhibit the ability to analyze the DNA. Therefore, DNA extraction methods have been developed to separate proteins and other cellular materials from the DNA molecules. In addition, the quantity and quality of DNA often need to be measured prior to proceeding further with analytical procedures to ensure optimal results.

There are three primary techniques for DNA extraction used in today's forensic DNA laboratory: organic extraction, Chelex extraction, and FTA paper (Figure 3.1). The exact extraction or DNA isolation procedure varies depending on the type of biological evidence being examined. For example, whole blood must be treated differently from a bloodstain or a bone fragment.

Organic extraction, sometimes referred to as phenol chloroform extraction, has been in use for the longest period of time and may be used for situations where either RFLP or PCR typing is performed. High molecular weight DNA, which is essential for RFLP methods, may be obtained most effectively with organic extraction.

The Chelex method of DNA extraction is more rapid than the organic extraction method. In addition, Chelex extraction involves fewer steps and thus fewer opportunities for sample-to-sample contamination. However, it produces single stranded DNA as a result of the extraction process and therefore is only useful for PCR-based testing procedures.

All samples must be carefully handled regardless of the DNA extraction method to avoid sample-to-sample contamination or introduction of extraneous DNA. The extraction process is probably where the DNA sample is more susceptible to contamination in the laboratory than at any other time in the forensic DNA analysis process. For this reason, laboratories usually process the evidence samples at separate times and sometimes even different locations from the reference samples.

A popular method for preparation of reference samples is to make a blood stain by applying a drop of blood on to a cotton cloth, referred to as a swatch, to produce a spot about 1 cm2 in area. Ten microliters of whole blood, about the size of a drop, contains approximately 70 000-80 000 white blood cells and should yield approximately 500 ng of genomic DNA. The actual yield will vary

ORGANIC

proteinase K

^^ Centrifuge

Phenol, chloroform, isoamyl alcohol

Phenol, chloroform, isoamyl alcohol

VORTEX

^ Centrifuge

TRANSFER aqueous (upper) phase to new tube

I^TE

i buffer

CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation)

CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation)

^^ Centrifuge

QUANTITATE DNA

PERFORM PCR

CHELEX

INCUBATE (ambient) ^^ Centrifuge

REMOVE supernatant

Chelex

QUANTITATE DNA

PERFORM PCR

FTA Paper

Apply blood to paper and allow stain to dry

PUNCH

WASH Multiple Times with extraction buffer

REMOVE supernatant

PCR Reagents

(NO DNA QUANTITATION REQUIRED)

PERFORM PCR

Figure 3.1

Schematic of commonly used DNA extraction processes.

with the number of white blood cells present in the sample and the efficiency of the DNA extraction process.

Extracted DNA is typically stored at —20°C, or even —80°C for long-term storage, to prevent nuclease activity. Nucleases are enzymes (proteins) found in cells that degrade DNA to allow for recycling of the nucleotide components. Nucleases need magnesium to work properly so one of the measures to prevent them from digesting DNA in blood is the use of purple-topped tubes containing a blood preservative known as EDTA. The EDTA chelates, or binds up, all of the free magnesium and thus prevents the nucleases from destroying the DNA in the collected blood sample.

Stuttering Simple Techniques to Help Control Your Stutter

Stuttering Simple Techniques to Help Control Your Stutter

Discover Simple Techniques to Help Control Your Stutter. Stuttering is annoying and embarrassing. If you or a member of your family stutters, you already know the impact it can have on your everyday life. Stuttering interferes with communication, and can make social situations very difficult. It can even be harmful to your school or business life.

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