Microbiology Lab Review: Chapter 18

Lab Review

Chapter 18: DNA Gel Electrophoresis

1. [CH-18-01] In what way are restriction enzymes specific?

Restriction enzymes cut DNA at specific palindromic sequences (same DNA sequence in 3’ to 5’ direction). The palindromic sequences vary depending on the specific restriction enzyme.

2. [CH-18-02] If you cut the same DNA with two different restriction enzymes separately would you get the same banding pattern? Explain the answer.

NO, the restriction enzymes each have their own specific recognized palindromic sequence. The resulting banding patterns would vary because of the different sizes of the fragments.

3. [CH-18-03] Explain how electrophoresis works –

DNA is separated in fragments by restriction enzymes. The fragments are then placed on a gel submerged in a buffer solution to be separated based on size using an electrical field (cathode – negative to anode – positive).

4. [CH-18-04] In your lab class, you have used gel electrophoresis to separate fragments of DNA. Can you use gel electrophoresis to separate proteins? Yes, or No, explain your answer –

YES, BUT since proteins are not always negatively charged they need to be treated with sodium dodecyl sulfate (unfolds proteins into linear and coats them with negative charges) so that they will move with the electrical field.

5. [CH-18-05] If the banding pattern from two DNA samples is the same, does it mean that the samples are identical? If your answer is no, then what would you have to do to show that they are identical? –

NO, the banding patterns rely heavily on the type of restriction enzyme used and the sample DNA. To get more accurate confirmations, the DNA samples have to be tested with multiple restriction enzymes and the banding patterns resulting form the multiple fragments have to be compared.

6. [CH-18-06] DNA electrophoresis is used for genomic typing of bacterial strains. What are serotyping and phage typing of bacterial strains? –

Bacterial serotyping – Classification of bacteria into specific strains depending on their DNA banding patterns.
Phage typing – Determining a specific strain based on the bacteriophage that targets and kills that strain.

7. [CH-18-07] What is the difference between sticky and blunt ends of DNA fragments formed during digestion by restriction enzymes? –

Sticky ends – Forms overhang (stretch of unpaired nucleotides), the orientation of new inserted DNA is specific b/c overhangs.
Blunt ends – NO overhang, DNA strand ends at a base pair, the potential for inserting DNA in wrong orientation.

8. [CH-18-08] Why is DNA moving toward anode during gel electrophoresis? –

DNA is negatively charged, because of oxygen bonded to phosphate, and move towards the (+) anode. Opposites attract (-) → (+).

9. [CH-18-09] Explain how the molecules are separated during gel electrophoresis.

Fragments of the DNA formed by restriction enzymes move towards the (+) anode. The larger fragments are slowed by resistance as they move through the pores of the cell, while the smaller fragments move more easily and get farther.

10. [CH-18-10] Why is DNA sample mixed with sample loading buffer before it is loaded on the gel?
    Sample loading buffer combines with DNA to increase the density of the sample and ensures that the DNA stays within the wells of the gel. Loading buffer also contains dyes that combine with the DNA fragments and allow us to visually “see” the fragments moving.