× Didn't find what you were looking for? Ask a question
Top Posters
Since Sunday
5
a
5
k
5
c
5
B
5
l
5
C
4
s
4
a
4
t
4
i
4
r
4
New Topic  
Salmams Salmams
wrote...
Posts: 693
Rep: 0 0
6 years ago
We can detect the SNP in the S allele because it destroys a DdeI restriction site, leading to DNA restriction fragment variation revealed by Southern blot analysis.
 
  Either two or three restriction sequences for the restriction endonuclease DdeI can occur near the -globin gene, depending on the allele. One DdeI restriction sequence is eliminated from the S allele by the SNP in the mutant allele because of a mutation in the double-stranded target sequence 5 - CTNAG - 3. Suppose the wild-type allele sequence is CCTGAG, while a newly identified SNP is CCTTAG. Would this same restriction fragment analysis technique be appropriate for detecting this particular SNP? What other technique(s) could you use to identify this particular mutation?
Read 22 times
1 Reply

Related Topics

Replies
wrote...
6 years ago
The DdeI target sequence is 5 - CTNAG - 3, where N indicates that any of the four nucleotides (A, T, C, or G) can occur in the middle of the 5-bp sequence as long as the variable nucleotide is flanked by CT and AG dinucleotide combinations. Thus, you would be unable to detect the difference between CCTGAG and CCTTAG by restriction fragment analysis. Several other techniques may help detect this particular mutation:
(1 ) You could design a molecular probe specific for the mutant sequence, although it may be difficult to detect a one-base-pair change in sequence. The probe would have to be very short, and the annealing conditions would need to be fairly stringent so that the probe would not anneal based solely on the homologous regions around the mutation.
(2 ) You could also look for changes at the protein level. This mutation occurred in an exon region, so you might expect to see a change in the amino acid sequence (depending on the translation frame). For instance, if GAG is one codon and TAG is the SNP's codon, you would see a Glu  Stop mutation and could look for the presence of a truncated protein in the mutant allele.
(3 ) Alternatively, it may be easiest to design a short primer upstream of this mutation and sequence the region directly using genomic DNA as a template for PCR (a technique that is described in detail in later chapters).
New Topic      
Explore
Post your homework questions and get free online help from our incredible volunteers
  1363 People Browsing
Related Images
  
 291
  
 175
  
 397
Your Opinion
Who's your favorite biologist?
Votes: 586