You were going to sequence a rice DNA fragment whose sequence was only know at one end, as shown below. 5’ AAACGATCGAGTCGCATCCAAAATCGATACCC—unknown region 3’ TTTGCTAGCTCTGCGTAGGTTTTAGCTATGGG—unknown region After several tries, you obtained a beautiful sequencing image as shown here: The worked out well partially because you had designed a primer for sequencing the unknown region according to the following guideline: Tm is 55 – 60°C. Ensures primer had a appropriate melting temperature for PCR ans sequencing. The GC content of the primer is the same as the genome/template (rice = 60%, human/Drosophila = 45-50%). A same nucleotide cannot be more than 2 in a row, e.g. CCC, GGGGG, AAA. The secondary structure of the primer must be none or weak. No primer dimers (The primer anneals to itself). 3’ end is the most important: it should not end in A, preferably ends in GG, GC, CG or CC This website can help you design the primer: http://www.oligoevaluator.com/OligoCalcServlet Question 1A. What is the primer sequence you designed? Please include the 5’ and 3’ ends. Question 1B. Why did you select the sequence as your primer?

Essentials of Pharmacology for Health Professions
7th Edition
ISBN:9781305441620
Author:WOODROW
Publisher:WOODROW
Chapter5: Abbreviations And Systems Of Measurement
Section: Chapter Questions
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You were going to sequence a rice DNA fragment whose sequence was only know at one end, as shown below.

5’ AAACGATCGAGTCGCATCCAAAATCGATACCC—unknown region

3’ TTTGCTAGCTCTGCGTAGGTTTTAGCTATGGG—unknown region

After several tries, you obtained a beautiful sequencing image as shown here:

The worked out well partially because you had designed a primer for sequencing the unknown region according to the following guideline:

  1.   Tm is 55 – 60°C.

Ensures primer had a appropriate melting temperature for PCR ans sequencing. 

  1.   The GC content of the primer is the same as the genome/template (rice = 60%, human/Drosophila = 45-50%).
  2.   A same nucleotide cannot be more than 2 in a row, e.g. CCC, GGGGG, AAA.
  3.   The secondary structure of the primer must be none or weak.
  4.   No primer dimers (The primer anneals to itself).
  5.   3’ end is the most important: it should not end in A, preferably ends in GG, GC, CG or CC

This website can help you design the primer: http://www.oligoevaluator.com/OligoCalcServlet

 

Question 1A. What is the primer sequence you designed?  Please include the 5’ and 3’ ends. 

Question 1B. Why did you select the sequence as your primer? 

ddA ddC ddG ddT
1.1
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Transcribed Image Text:ddA ddC ddG ddT 1.1 }}}] ”ו
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