7.3.4 Southern Blotting

Question 1

southern blotting

Answer 1

• a laboratory technique for DNA visualization that was developed by E. M. Southern in 1975.
  • The process of Southern blotting involves the transfer of DNA segments from an electrophoresis gel to a sheet of nylon paper (or nitrocellulose membrane) and hybridizing the segments with a radioactive probe.
• By taking an autoradiogram of the nylon paper, a photographic negative will reveal locations of the bands of DNA that contain the target sequence.
  • Southern blotting is a procedure used in conjunction with many other techniques in biotechnology, such as RFLP analys is and DNA fingerprinting.

Question 2

note

Answer 2

• Specific DNA segments within an electrophoresis gel can be visualized with the Southern blotting technique.
• Suppose that you want to find out which of the bands in the gel to the left contain the sequence AAATTT.
• Southern blotting can be used to determine which of the bands in the gel to the left contain the sequence AAATTT.
• The gel is placed into a container that has special buffers and chemicals that are able to denature the DNA. The gel is then placed onto a platform that is covered with filter paper within the container. A positively charged piece of membranous paper (nylon or nitrocellulose) is placed on top of the gel. A stack of paper towels is placed on top of the charged paper
  and weight is applied.
• As solution is drawn up through the layers of paper, DNA is transferred from the gel to the charged nylon membrane. The DNA within the membrane is denatured by the solution, which results in bands of single stranded DNA positioned as they were in the gel.
• To visualize the specific sequence of DNA, the membrane is placed in a plastic bag containing a radioactive DNA probe. The probe, with a sequence complimentary to the target sequence (in this case TTTAAA), is allowed to hybridize with the DNA. The membrane is then rinsed to wash off excess unhybridized probe.
• In a process called autoradiography, the membrane is
  exposed to film paper. Wherever the radioactive probe
  hybridizes with DNA containing a complimentary sequence, there will be a corresponding image on the film paper.

Question 3

Why can Southern blotting be used to analyze entire chromosomes, while simple gel electrophoresis is only useful for much smaller DNA fragments?

Answer 3

• Southern blot only visualizes the DNA sequences of interest

Question 4

Southern blotting is

Answer 4

• effective even with large amounts of DNA such as an entire chromosome.
• a method of detecting specific DNA sequences.
• a technique named for its creator, E.M. Southern

Question 5

The final result of Southern blotting is

Answer 5

• a film with bands representing only those fragments containing the nucleotide sequence of interest

Question 6

The basis for the detection of specific sequences using the Southern blot technique is

Answer 6

• nucleic acid hybridization.

Question 7

Place the following steps for Southern blot in the proper order.

i. blot with filter paper
ii. cut DNA with restriction enzyme
iii. autoradiography
iv. hybridize with radioactive (or fluorescent) probes
v. electrophoresis

Answer 7

• ii, v, i, iv, iii