Dithiothreitol, Saifool and Sodomy 2.0: We can see (obviously) that the Empirical Derivations of the facts that the evidence has been fabricated. Without the implementation of DEP – Differential Extraction Process and The Usage of a small-molecule redox reagent known as Cleland’s reagent, The DNA that “claimed” to be extracted from the sperm is false and it has been fabricated from other part of human being – Daubert Standard

Differential extraction

From Wikipedia, the free encyclopedia

Differential extraction refers to the process by which the DNA from two different types of cells can be extracted without mixing their contents. The most common application of this method is the extraction of DNA from vaginal epithelial cells and sperm cells from sexual assault cases in order to determine the DNA profiles of the victim and the perpetrator. Its success is based on the fact that sperm cells have protein disulfide bonds in their outer membrane which makes them more resilient to extraction than epithelial cells.

After determining that sperm cells are present (typically through staining and light microscopy) in a vaginal/rectal sample, the subject’s epithelial cells are lysed by a standard DNA extraction method, like a phenol/chloroform extraction and their DNA extracted through normal means. The epithelial DNA in solution is removed and saved, while the sperm cells are still intact. Differential extraction uses a chemical called dithiothreitol (DTT) to disrupt the sulfur bonds in the coating of the sperm cell in order to extract its DNA. Once the sperm’s outer membrane has been breached, it is prone to standard DNA extraction methods. This creates two different DNA fractions from one sample, hopefully that of the victim and that of the perpetrator.

http://en.wikipedia.org/wiki/Differential_extraction

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Dithiothreitol (DTT) is the common name for a small-molecule redox reagent known as Cleland’s reagent. DTT’s formula is C₄H₁₀O₂S₂ and the molecular structure of its reduced form is shown at the right; its oxidized form is a disulfide-bonded 6-membered ring (shown below). Its name derives from the four-carbon sugar, threose. DTT has an epimeric (‘sister’) compound, dithioerythritol (DTE).

Contents [hide] 1 Reducing agent 2 Applications 3 Properties 4 References

[edit] Reducing agent

DTT is an unusually strong reducing agent, owing to its high conformational propensity to form a six-membered ring with an internal disulfide bond. It has a redox potential of -0.33 V at pH 7. The reduction of a typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions and is illustrated below. The intermediate mixed-disulfide state is unstable (i.e., poorly populated) because the second thiol of DTT has a high propensity to close the ring, forming oxidized DTT and leaving behind a reduced disulfide bond. The reducing power of DTT is limited to pH values above ~7, since only the negatively charged thiolate form -S^– is reactive (the protonated thiol form -SH is not); the pKa of the thiol groups is 9.2 and 10.1.

Reduction of a typical disulfide bond by DTT via two sequential thiol-disulfide exchange reactions.

[edit] Applications

A common use of DTT is as a reducing or “deprotecting” agent for thiolated DNA. The terminal sulfur atoms of thiolated DNA have a tendency to form dimers in solution, especially in the presence of oxygen. Dimerization greatly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold in biosensors. Typically DTT is mixed with a DNA solution and allowed to react, and then is removed by filtration (for the solid catalyst) or by chromatography (for the liquid form). The DTT removal procedure is often called “desalting.”

DTT is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins. However, even DTT cannot reduce buried (solvent-inaccessible) disulfide bonds, so reduction of disulfide bonds is sometimes carried out under denaturing conditions (e.g., at high temperatures, or in the presence of a strong denaturant such as 6 M guanidinium hydrochloride, 8 M urea, or 1% sodium dodecylsulfate). Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT.

DTT can also be used as an oxidizing agent. Its principal advantage is that effectively no mixed-disulfide species are populated, in contrast to other agents such as glutathione. In very rare cases, a DTT adduct may be formed, i.e., the two sulfur atoms of DTT may form disulfide bonds to different sulfur atoms; in such cases, DTT cannot cyclize since it has no remaining free thiols.

[edit] Properties

Due to air oxidation, DTT is a relatively unstable compound whose useful life can be extended by refrigeration and handling in an inert atmosphere. Since protonated sulfurs have lowered nucleophilicities, DTT becomes less potent as the pH lowers. Tris(2-carboxyethyl)phosphine HCl (TCEP hydrochloride) is an alternative which is more stable and works even at low pH.

[edit] References

1. ^ Merck Index, 11th Edition, 3382.

• Cleland, W.W. (April 1964). “Dithiothreitol, A New Protective Reagent for SH Groups”. Biochemistry 3: 480–2. doi:10.1021/bi00892a002. PMID 14192894.
• Ruegg, UT; Rudinger, J. (1977). “Cleavage of disulfide bonds in proteins”. Methods Enzymol 47: 111.

http://en.wikipedia.org/wiki/Dithiothreitol

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How is it that three cotton swabs retrieved from the rectum of sodomy complainant Mohd Saiful Bukhari Azlan showed no sign of degradation when tested some 100 hours later?

This is the issue that is puzzling Australian DNA expert Dr Brian McDonald.

Describing the findings from the three swabs marked B7, B8 and B9 from the high and low rectal areas of the complainant as “pristine”, McDonald (right) told the Sodomy II trial of Opposition Leader Anwar Ibrahim that this was “inconsistent with the history”.

“This is because there is no sign of degradation and the samples are pristine,” he said about the three samples taken from Saiful’s rectum 56 hours after the alleged sodomy took place between 3.01pm and 4.30pm on June 26, 2008.

Saiful was examined by four doctors at Hospital Kuala Lumpur between 9pm on June 28 and midnight on June 29, 2008.

Investigating officer Supt Jude Blacious Pereira had earlier testified that the samples were kept in a drawer in his air-conditioned office and handed over to chemist Dr Seah Lay Hong at around 7pm on June 30, 2008.

Yesterday, McDonald, 60, told the Kuala Lumpur High Court that the samples should ideally be kept in a freezer at minus 20 degrees Celsius.

Questioned on the three samples by defence counsel Ram Karpal Singh, McDonald said the ‘Male Y’ DNA was prevalent in the low rectal swabs.

However, the witness noted, Saiful’s DNA was prevalent in the high rectal swab.

“Hence, I cannot discount the possibility that Saiful’s semen or sperm could have been up in the high rectal area, based on the tests,” McDonald said.

“Furthermore,” he added, “the three samples from the swab are pristine, although they should have degraded under the circumstances.”

Another Australian expert witness, Dr David Wells, had testified earlier that he would not take samples from a sexual assault victim 36 hours after the attack.

McDonald had also testified that such a sample would be deeply degraded after 56 hours.

Saiful’s semen sperm in anus?

McDonald also explained that chemists Dr Seah Lay Hong was guilty of “guessing” in treating Saiful’s sample, as there was no indication in her report where the sample originates and that he did not use the same numbering system employed by the doctors.

“We have to assume where the swab came from as this is not reflected in Seah’s (right) report,” he said, adding Seah should verify where it came from in sodomy cases.

“It could come from a cigarette butt. Seah did not provide the information in the report. She has not reported anything on the sample whether it came somewhere or another.”

McDonald said Seah did not properly do the differential extraction process (DEP) test from the samples taken to resolve the issue of sperm and non-sperm cells.

This, he said, has to be done again and again until what is left are sperm and not other contaminants.

He said this has resulted in findings of Saiful’s DNA in the rectum.

“That is the case of the complainant’s semen is in his rectum,” he said, adding one cannot assume there is Saiful’s sperm is in his own rectum initially but this had to be assumed following the results of the test.

“I would say Seah’s evidence is a guess.”

Ram: Why is that?

McDonald: Because she did not slide on the receptacle and put it under a microscope to verify. That is only way to determine and if there is further, the process has to be repeated again to remove the contaminants.

Ram: Is it possible that there is Saiful’s sperm in his anus.

McDonald: If she had done it properly, we can cut out on such a possibility. The sample taken cannot be relied on because (it contain Saiful’s DNA).

Ram: Can we conclusively say that Saiful’s sperm is not in his anus?

McDonald: No, she is guessing that there are other cells. She did not do the DEP test properly. The Male Y (allegedly Anwar’s DNA) is predominant in B7 the low rectal swab. However, on the B8 (high rectal), Saiful’s DNA was predominant.We cannot discount Saiful’s sperm was up in his own anus, based on the result.

Anwar Ibrahim is charged with sodomising Saiful at the Desa Damansara condominium between 3.01pm and 4.30pm on June 26, 2008.

The trial continues from Sept 19 to 23 with McDonald’s testimony.

http://anwaribrahimblog.com/2011/08/26/pristine-condition-of-rectal-swabs-puzzles-expert/

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