1b) If you perform the same experiment using DNA extracted from Thermus aquaticu

Question

1b) If you perform the same experiment using DNA extracted from Thermus aquaticus, an unusual bacterial species that grows in hot springs (average temperature 93.5°C), you will find that all of the DNA fragments form a single band at a single density in the density gradient, which gives a single peak when the results are graphed. Why do the DNA fragments from T. aquaticus give a different result in this experiment than the DNA fragments from fossa genomic DNA?

1c) T. aquaticus genomic DNA is 33.7% guanosine nucleotides. What fraction of the DNA is adenosine nucleotides?

1d) If you took the same randomly generated 500 bp fragments of fossa DNA that you used in the equilibrium density gradient centrifugation experiment described in part a of this question and used them as a sample in agarose gel electrophoresis, how many bands would you expect to find in the gel when you turned off the current and stained the gel with ethidium bromide? Briefly explain why you would predict that number of bands.

1e) DNA sequencing of the fossa genome reveals that ~15% of the genome is composed of highly repetitive sequences, ~22% of the genome is moderately repetitive sequences, and the remaining 63% of the genomic DNA is low copy number and unique sequences. If you took the random 500 bp fragments of fossa genomic DNA that you generated in part a of this question, denatured them by heating them to 95°C, and allowed them reanneal at 60°C, the highly repetitive fragments would reanneal much more quickly than the low copy number and unique fragments. Briefly explain, in your own words, why this is the case.The fossa is a unique carnivore found only in Madagascar. If you extract genomic DNA from fossa cells, randomly shear the DNA into 500 bp fragments, and use those DNA fragments in an equilibrium density gradient centrifugation experiment, you get results similar to those shown below. What can you conclude about the base composition and base distribution of G-C base pairs in the fossa genomic DNA from this result? Briefly explain how or why you would reach those conclusions.  

Explanation / Answer

#1a)

In density gradient centrifugation, under the influence of centrifugal force, DNA fragment move through a density gradient and be suspended at a point in which the density of the DNA fragment equals the surrounding medium.

As here, the fragments are sized almost equally ~500bp, the density is based on its GC content % making a DNA fragment denser if its GC content is higher.

The genome of Fossa (eukaryote) contains 75% of chromosomal DNA with a sharp high peak and the smaller peaks with 10 and 15 percent of the DNA fragments are short repetative or satellite DNA having less and more GC content than the chromosomal DNA respectively.

#1b)

Thermus aquaticus, an unusual bacterial species that grows in hot springs is a prokayote which has satellite DNA features in plasmid DNA which is independent of the chromosomal DNA/ genomic DNA. Thus, the prokaryotic (T. aquaticus) genome would show a single sharp peak for chromosomal DNA unlike the genomic DNA of an eukaryote (Fossa).

#1c)

Using Chargaff's rule of base pairing:

A+T = G+C also, G=C, A=T

Here, G=33.7%, therefore, C= 33.7%

Hence, T= (100-(2 x 33.7))/ 2 = 16.3 %

#1d)

Gel electrophoresis separates DNA according to its size. Thus, the randomly generated fragment will show only one thick band at around 500bp because the complete genomic DNA is being fragmented around 500 basepairs (bp).

#1e)

High temperature denatures DNA by breaking the hydrogen bonds in between the nucleotides of the two strands. We know that guanine forms three hydrogen bonds with cytosine and adenine forms two hydrogen bonds with thymine. Thus, the GC pair will need higher temperature to denature and more time to re-anneal back into double strand.

If reannealing of the the highly repetitive fragments is happening more quickly than the low copy number and unique fragments. Then its directly infers that the GC content of highly repetitive fragments is higher than the low copy number and unique fragments.

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