Scientists decided to study, in vitro, the activity of the eukaryotic DNA polymerase Pol e. 1)...

Scientists decided to study, in vitro, the activity of the eukaryotic DNA polymerase Pol e.
1) In a first experiment, they apply a solution of Pol e on top of a chromatography column containing heparin. They collect the solution that elutes from the bottom of the column (flow-through). Their analysis reveals that the flow-through does not contain any protein.
What is the property of heparin demonstrated by this experience?

2) They designed a second experiment including 4 steps.
Step 1: A long single stranded DNA (M13) was incubated with a radiolabeled primer (P) with a sequence complementary to a short region of M13.
Step 2: The DNA/P complex was then incubated with the DNA polymerase, the 4 types of deoxyribonucleoside 3-phosphate (dNTPs), with or without heparin.
Step 3: After incubation for different length of times (0, 1, 2, and 4 minutes) the DNA polymerase is inactivated, the DNA is extracted and separated by agarose gel electrophoresis.
Step 4: The radioactive DNA was visualized by autoradiography.

Scientists compared the activity of two DNA polymerase: and Intact polymerase (Pol e WT) and, a mutant polymerase lacking a portion of the protein (Pol e D). Not that the deletion does not affect the catalytic activity of DNA Pol e.
The results of the experiments are listed in the figure below.

a) What does the 31 nucleotides (nt) fragment represent? Be specific
b) Why do you think the 31 nt fragment was not detected when Pol e WT was incubated with heparin?

c) By comparing the data for Pol e WT and Pol e D and based on what you know about this polymerase, could you identify the domain of Pol e missing in Pol e D Briefly explain?

Expert Solution

1)It shows that the heparin has the property to interfere in the process of replication . The concentration of the heparin determines the activity of DNA dependent RNA polymerase . The analysis reveals that the flow through does'nt contain any protein because the activity of the polymerase enzyme is inhibited and thus polymerase enzyme do not produce any RNA hence , no protein synthesis , therefore the amplification of the DNA of the stored blood is very difficult as it contains a particular concentration of heparin . To overcome this certain methods such as gllustra nucleon genomic method is used for the extraction of the DNA from the blood and this contains very low concentration of heparin and decrease the inhibitory effect. Phenyl / chloroform purification is used for the purification of blood sample . Taq polymerase enzyme which is produced by the bacteria thermus aquaticus is a heat stable enzyme used in the amplification of DNA.

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