Updated: 3/8/2020

Blotting

0%
Topic
Review Topic
0
0
N/A
N/A
Questions
3
0
0
0%
0%
Evidence
1
0
0
0%
0%
Videos / Pods
2
Topic
Blotting Procedures
  • This image depicts the steps of a Southern blot.  First, the DNA is digested by a restriction enzyme.  Next, the digested DNA is separated by size by gel electrophoresis.  The DNA fragments are then transferred to a nitrocellulose paper.  Finally, a DNA tag is hybridized to the sequence of interest and is validated. Function
    • probe for specific substance in solution
      • Southern blot - used to analyze DNA
        • normally used to examine the presence of a particular DNA sequence
        • remember: Southern Dixieland
      • Northern blot - used to analyze RNA
        • normally used to examine gene expression
      • Western blot (immunoblot) - used to analyze protein
  • Process
    • run a gel electrophoresis to separate the components of the solution
    • bands are transferred ("blotted") to a filter/membrane
    • radiolabeled or fluorescently labeled probe is incubated with the membrane
      • Southern blot - 32P-DNA 
      • Northern blot - 32P-DNA
      • Western blot - enzyme-linked or 131I antibody
    • specific probe binds with high specificity to DNA/RNA segment or protein of interest
    • membrane is visualized under conditions to illuminate the probe
  • Clinical use
    • restriction length polymorphism (RFLP) 
      • can be used for genetic testing
      • based on the principle that individuals have unique/heritable variations in RFLPs
        • loss or gain of a restriction site changes the fragment lengths after digestion by an endonuclease
        • variation provided in part by variable number tandem repeats (VNTRs)
      • Southern blot used to visualize results
      • ex.) testing for sickle-cell anemia
        • wild type (wt) gene contains a restriction site in the gene fragment
        • sickle cell (sc) gene lacks this restriction site
        • the diagnostic results following a Southern blot
          • wt homozygote would have only medium and small fragments
          • heterozygote would have large, medium and small fragments
          • sc homozygote would have only large fragments
    • gene expression profiling
      • measure whether a particular region of DNA is being expressed
      • Northern blot used to visualize results
      • ex.) testing for fragile X syndrome
        • expression of FMR1 gene
        • trinucleotide repeat disorder

Please rate topic.

Average 5.0 of 3 Ratings

Questions (3)
Question locked
Sorry, this question is for
PEAK Premium Subscribers only
Upgrade to PEAK

(M1.BC.15.74) A scientist wants to determine if a specific fragment is contained within genome X. She uses a restriction enzyme to digest the genome into smaller fragments to run on an agarose gel, with the goal of separating the resulting fragments. A nitrocellulose blotting paper is then used to transfer the fragments from the agarose gel. A radiolabeled probe containing a complementary sequence to the fragment she is searching for is incubated with the blotting paper. Which of the following is the RNA equivalent of this technique?

QID: 106644
1

Southern blot

15%

(35/231)

2

Northern blot

68%

(158/231)

3

Western blot

7%

(17/231)

4

qPCR

2%

(5/231)

5

RT-PCR

6%

(13/231)

M 1 D

Select Answer to see Preferred Response

Question locked
Sorry, this question is for
PEAK Premium Subscribers only
Upgrade to PEAK
Evidence (1)
VIDEOS & PODCASTS (2)
EXPERT COMMENTS (16)
Private Note