You are provided with a concentrated, purified, nucleic acid sample containing a

Question

You are provided with a concentrated, purified, nucleic acid sample containing a single DNA molecule. (15 points) 9. (a) Describe how electrophoresis could have been used to prepare this sample and specify on factors affecting electrophoretic mobility. Could the size of the DNA molecule be estimated using this technique? (b) How would you go about determining if the DNA molecule was single-stranded or double-stranded? Be specific with regards to your proposed experiment(s) and the properties/data you are monitoring. (e) You have decided to perform digestion and obtained the following results: e Fragment Size (kb) Enzyme EcoRI Hpall HindIll EcoRI+ Hpall 1.3, 0.8, 0.5 EcoRI+Hindill 0.6,0.7, 1.3 1.3, 1.3 2.6 2.6 Is the original DNA molecule circular or linear? Prepare a map of the restriction enzyme sites indicating the distance between them. a. b.

Explanation / Answer

Answer:

a) The DNA sample can be analyzed through electrophoresis after performing polymerase chain reaction using DNA primers. This is followed by mixing the amplified product with required concentrations of Sybr green or Sybr gold that can bind to single-stranded DNA samples. DNA is then loaded onto the wells of gel and the apparatus is connected to power. Since the DNA is negatively charged, the samples move towards the anode. In agarose gel electrophoresis, samples move based on its size. To identify the size of DNA molecule, a DNA ladder is also loaded.

b) To determine the single stranded or double stranded DNA structure, the DNA is run on gel and then visualized for bands. Single stranded DNA move faster than double stranded due to their structure.

c) a. Original DNA molecule was circular that was linearized using restriction enzyme digestion.

b. EcoRI : <EcoRI---------------------EcoRI><--------------->HpaII------<HindIII>-------(Starting EcoRI site)

<----------1.3-----------------------><----0.5------><0.8-----------------------------------------------.

<-----------------0.7----------------->

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