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Anomaly Physical Evidence Investigation Group & DNA Investigations |
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IUR F SPRING 1999 4 Peter Khoury pt 12
Those two internal DNA primers extend over locations 16,190–16,209 (upper strand) or 16,271–16,294 (lower strand) within the hypervariable region itself. Thus, when primers 15,993–16,022 (upper) and 16,271–16,294 (lower) are used as a pair in PCR, they will 16,108–T 16,129–A 16,162–G 16,172–C 16,172–C 16,162–G 16,129–A 16,108–T yield a mitochondrial DNA fragment spanning the left-hand side of the hypervariable region, as a product of size 302 base pairs.
Similarly, when primers 16,190–16,209 (upper) and 16,401-16,430 (lower) are used as a pair, they will yield a 241 bp product spanning the right hand side of the hypervariable region (see Mitomap for details). Each PCR reaction was carried out under standard conditions using 1.5 mM magnesium chloride, and for 30 to 36 cycles of denaturing at 95° C., extending at 72° C., and annealing at 57° C.
All reactions were checked by agarose gel electrophoresis after just 30 cycles, to see whether the amplification had proceeded efficiently, and then run for another six cycles if only small amounts of product were observed. The background contamination of control samples not containing any hair (by other DNA in the laboratory) was never more than 10% of the product. Magnified hair sample showing optical transparency and pronounced mosaic structure of anomalous hair. The circles of lights are reflections.
Photo taken from video. B. Chalker/APEG. Once PCR products from both the left- and right-hand sides of the hypervariable region were obtained, in quanti-ties of at least 100 nanograms, each DNA band was purified by excision from an ethidium bromide stained agarose gel, following several hours of electrophoresis. Next, each excised band was treated with high salt and silica beads, to extract the DNA.
Finally, each amplified DNA fragment of size 302 or 241 base pairs was trimmed with Pfu polymerase to create blunt ends for cloning, then treated with polynucleotide kinase to add 5'-phosphates. RESULTS Four PCR products were obtained in total: two from the hair of the tall blonde female (both left and right), two from Peter Khoury (both left and right), but none from his wife, whose hair may have been chemically treated so as to make recovery of DNA difficult (and who never came into direct contact with the alien hair).
These four amplified DNA fragments were then cloned into a commonly used plasmid vector. Four full libraries of cloned sequences were obtained, by transformation of the four ligation reactions into E. coli, followed by multiple preparations of plasmid DNA on a small scale. Roughly 6–12 clones were obtained from each amplified DNA fragment as a fairly substantial library by which to assess whether the amplified DNA might be pure and authentic, or else contain impurities due to contaminating DNA.
Screening of these libraries using the dideoxy method for just one nucleotide showed that all four libraries contained pure and homogenous DNA, without any sequence impurities to a 90% level. Some clones were of reduced length, but these turned out to be end deletions as produced in the PCR step or subsequent manipulation. Full sequence analysis of all four bases G, A,T and C using the dideoxy method (Sequenase Version 2.0) showed that all clones from the young man’s hair matched closely the human consensus from Mitomap, which is European in nature.
Thus, clones from his hair did not show any systematic deviation from the consensus in any of 380 locations spanning the whole hypervariable region 16,023–16,400, apart from a few common C-to-T or A-to-G heteroplasmies at 16,189, 16,270, 16,312 or 16,362. By way of contrast, all clones as amplified from the hair of the tall blonde female show five consistent substitutions from the human consensus.
That consensus among human geneticists is based on the typical white European DNA sequence. These are all C/T or A/G transitions, and are located at 16,108 (C to T), 16,129 (G to A), 16,162 (A to G), 16,172 (T to C), and 16,304 (T to C). Three of those transitions at 16,129, 16,172, and 16,304 seem fairly common among human racial types, whereas the two at 16,108 and 16,162 seem quite rare. Thus, mitochondrial DNA analysis of the hair shaft from a reportedly tall, blonde alien female shows that she is biologically close to normal human genetics, but of an unusual racial type. (Geneticists consider that there are two major racial types, based on DNA analysis: Africans and white Asians.)
By comparison, Neanderthal man differs from modern humans at 27 locations in the same DNA, while the chimpanzee differs from humans at 55 (see M. Krings, et al., “Neanderthal DNA Sequences and the Origin of Modern Humans,” Cell 90 (1997): 19–30), but the DNA from the tall blonde differs from the chimpanzee at 60 locations.
One might expect from the physical description of that tall blonde female, as well as from the possible sexual nature of the event, that such a tall blonde female might represent just some strange human racial type.Strange Evidence : [01] [02] [03] [04] [05] [06] [07] [08] [09] [10] [11] [12] [13] [14]
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