DNA Extraction

DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic steps in a DNA extraction:

1. Breaking the cells open to expose the DNA within, such as by grinding or sonicating the sample.
2. Removing membrane lipids by adding a detergent.
3. Precipitating the DNA with an alcohol — usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet on centrifugation. This step also removes alcohol-soluble salt.

Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+. This stops dnase enzymes from degrading the DNA.

  Subscribe in a reader

Cellular and histone proteins bound to the DNA can be removed prior to its precipitation either by adding a protease or having prior to precipitation, precipitating with sodium or ammonium acetate, or extracting with a phenol-chloroform mixture.

If desired, the DNA can be resolubilized in a slightly alkaline buffer.

Detecting DNA

A diphenylamine (DPA) indicators will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95oC) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.

Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nm, and aromatic proteins absorbs UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.

Using the Southern blot technique this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison and identification.

Nuclear Transfer

Nuclear Transfer is a form of cloning. The steps involve removing the DNA from an oocyte(unfertilized egg), and injecting the nucleus which contains the DNA to be cloned. In rare instances, the newly constructed cell will divide normally, replicating the new DNA while remaining in a pluripotent state. If the cloned cells are placed in the uterus of a female mammal, a cloned organism develops to term in rare instances. This is how Dolly the Sheep and many other species were cloned. Alternatively, if cells are extracted from the cloned cells during very early embryonic stages (blastocyst or morula), embryonic stem cells can be created. These cells can be grown in laboratories indefinitely and can theoretically be made into any of the 200+ cell types in the mammalian body, and thus are an extraordinary tool for biologists as well as a therapeutic agent with the potential to treat currently untreatable medical conditions.

Tools & Reagents

Nuclear transfer is a delicate process that is a major hurdle in the development of cloning technology! Materials used in this procedure are a microscope, a holding pipette (small vacuum) to keep the oocyte in place, and a micropipette (hair-thin needle) capable of extracting the nucleus of a cell using a vacuum. For some species, such as mouse, a drill is used to pierce the outer layers of the oocyte.

Various chemical reagents are used to increase cloning efficiency. Microtubule inhibitors, such as nocodazole, are used to arrest the oocyte in M phase, during which its nuclear membrane is dissolved. Chemicals are also used to stimulate oocyte activation.

Evolutionary significance of Human Chromosome 2

All apes apart from man have 24 pairs of chromosomes. There is therefore a hypothesis that the common ancestor of all great apes had 24 pairs of chromosomes and that the fusion of two of the ancestor's chromosomes created chromosome 2 in humans. The evidence for this hypothesis is very strong.

The Evidence

Evidence for fusing of two ancestral chromosomes to create human chromosome 2 and where there has been no fusion in other Great Apes is:

1) The analogous chromosomes (2p and 2q) in the non-human great apes can be shown, when laid end to end, to create an identical banding structure to the human chromosome 2.

2) The remains of the sequence that the chromosome has on its ends (the telomere) is found in the middle of human chromosome 2 where the ancestral chromosomes fused.


  Subscribe in a reader

3) the detail of this region (pre-telomeric sequence, telomeric sequence, reversed telomeric sequence, pre-telomeric sequence) is exactly what we would expect from a fusion.
4) this telomeric region is exactly where one would expect to find it if a fusion had occurred in the middle of human chromosome 2.

5) the centromere of human chromosome 2 lines up with the chimp chromosome 2p chromosomal centromere.

6) At the place where we would expect it on the human chromosome we find the remnants of the chimp 2q centromere .

Not only is this strong evidence for a fusion event, but it is also strong evidence for common ancestry; in fact, it is hard to explain by any other mechanism.


Centromere evidence

Let us re-iterate what we find on human chromosome 2. Its centromere is at the same place as the chimpanzee chromosome 2p as determined by sequence similarity. Even more telling is the fact that on the 2q arm of the human chromosome 2 is the unmistakable remains of the original chromosome centromere of the common ancestor of human and chimp 2q chromosome, at the same position as the chimp 2q centromere (this structure in humans no longer acts as a centromere for chromosome 2.

Referred
http://www.evolutionpages.com/chromosome_2.htm