Question

1. A 2 kb fragment of DNA was cut by EcoRI and BamHI and then analyzed by gel electrophoresis. The following fragment sizes were produced:

EcoRI: 900, 700, 400

BamHI: 1100, 900

In order to map the location of each restriction site, each fragment was then digested with the other two restriction enzymes. The following was obtained:

BamHI-Treated	EcoRI-Treated	Original Fragment Size
700, 200	-	900	EcoRI
700	-	700
400	-	400
-	700, 400	1100	BamHI
-	700, 200	900

Based on the fragment size analysis performed above, construct the map of restriction sites for this 2 kb fragment of DNA.

2.     Most restriction enzymes (RE’s) were isolated from bacteria. Since these bacteria require an intact (i.e. undigested) genome in order to survive, what mechanism protects the genomic DNA of bacteria from digestion by their own restriction enzymes?

3. Today we examined the pattern of genomic DNA when it is cut by a restriction enzyme. One of the enzymes, EcoRI, recognizes a specific 6 bp sequence. How often would you expect this enzyme to cut a long stretch of DNA? About how many fragments does an EcoRI digest produce on the calf genome (Bos taurus, 3 x 10⁹ basepairs in length)? Show all work.

4.   There are two principles underlying gel electrophoresis: charge, and the use of a gel matrix. Explain why both of these are necessary to separate DNA by size. Why is charge alone insufficient? Why is a gel matrix without charge insufficient?

5.     Agarose gel electrophoresis can size fractionate DNA fragments. If a 1000 bp linear DNA fragment and plasmid DNA 1000 bp in length are both run on the same gel, will they appear as the same size? Why or why not?

6.    Today we amplified 50 ng of Bos taurus (calf) DNA by PCR. This amount of DNA contains about 15,000 molecules of the insulin gene [50 ng DNA= 2.5 x 10^-20 mol; (2.5x10^-20 )x(6.023×10²³) = 1.5 x 10⁴ molecules]. We performed PCR for 35 cycles to amplify the amount of this gene.

      a. What is the theoretical fold amount of DNA amplified by 35 cycles of PCR (remember the 2^N formula)?

      b. How many molecules of the insulin gene would therefore be present after PCR?

7.   In theory, PCR exponentially amplifies a DNA fragment for x number of cycles. However, the concentration of PCR products will usually never reach the theoretical amount of amplification. Why do you think this is the case?

Answer 1

EcoRI: 900, 700, 400

BamHI: 1100, 900

BamHI-Treated	EcoRI-Treated	Original Fragment Size
700, 200	-	900	EcoRI
700	-	700
400	-	400
-	700, 400	1100	BamHI
-	700, 200	900

EcoRI

EcoRI

BamHI

700bp

200bp

700bp

400bp

2. Bacteria protects its own DNA from restriction enzymes by the process of methylation. It methylates specific nucleotides at the restriction sites. Restriction enzymes can digest only unmethylated DNA. Thus methylation protects bacterial DNA from restriction enzyme digestion.
3. EcoRI cuts the DNA at a 6 base pair long sequence. DNA has 4 different nucleotides. Therefore 4⁶ combinations are possible for 6 base pair long sequence.

4⁶ = 4096

Therefore, EcoRI will cut a DNA at a 6 base pair long sequence every 1 in 4096 times.

Calf genome = 3 x 10⁹ base pairs

EcoRI site is found every 4096 base pairs

Number of EcoRI sites in calf genome = 3 x 10⁹ / 4096 = 732421.8

Number of EcoRI fragments = 732422

4. Charge separates the DNA molecules based on its total charge. DNA is negatively charged and will move towards the positively charged anode. Depending on the size of DNA the negative charge varies. smaller DNA will have smaller negative charge.

The gel matrix separates the DNA based on the size. Smaller DNA moves faster towards the anode while the larger DNA moves slowly.

Charge alone is insufficient because you need a base in which to load the gel and visualise the separation of DNA fragments based on size.

A gel matrix without charge is insufficient because without the application of charge the DNA will not move towards the anode at different speed, resulting in DNA separation.

5. No. they will appear as different sizes. Linear DNA will have more friction in the gel matrix and hence will move slower than the plasmid DNA of same size which will be circular.