Question

. Find the all Restriction enzyme sites present in the following sequence and design primers for PCR.

gctcccggct tagaggacag cggggaaggc gggcggtggg gcagggggcc tgaagcggcg gtaccggtgc tggcggcggc agctgaggcc ttggccgaag ccgcgcgaac ctcagggcaa gatgcttgga accggacctg ccgccgccac caccgctgcc accacatcta gcaatgtgag cgtcctgcag cagtttgcca gtggcctaaa gagccggaat gaggaaacca gggccaaagc cgccaaggag ctccagcact atgtcaccat ggaactccga gagatgagtc aagaggagtc tactcgcttc tatgaccaac tgaaccatca catttttgaa ttggtttcca gctcagatgc caatgagagg aaaggtggca tcttggccat agctagcctc ataggagtgg aaggtgggaa tgccacccga attggcagat ttgccaacta tcttcggaac ctcctcccct ccaatgaccc agttgtcatg gaaatggcat ccaaggccat tggccgtctt gccatggcag gggacacttt taccgctgag tacgtggaat ttgaggtgaa gcgagccctg gaatggctgg gtgctgaccg caatgagggc cggagacatg cagctgtcct ggttctccgt gagctggcca tcagcgtccc taccttcttc ttccagcaag tgcaaccctt ctttgacaac atttttgtgg ccgtgtggga ccccaaacag gccatccgtg agggagctgt agccgccctt cgtgcctgtc tgattctcac aacccagcgt gagccgaagg agatgcagaa gcctcagtgg tacaggcaca catttgaaga agcagagaag ggatttgatg agaccttggc caaagagaag ggcatgaatc gggatgatcg gatccatgga gccttgttga tccttaacga gctggtccga atcagcagca tggagggaga gcgtctgaga gaagaaatgg aagaaatcac acagcagcag ctggtacacg acaagtactg caaagatctc atgggcttcg gaacaaaacc tcgtcacatt acccccttca ccagtttcca ggctgtacag ccccagcagt caaatgcctt ggtggggctg ctggggtaca gctctcacca aggcctcatg ggatttggga cctcccccag tccagctaag tccaccctgg tggagagccg gtgttgcaga gacttgatgg aggagaaatt tgatcaggtg tgccagtggg tgctgaaatg

I was not given any specific restriction enzymes for this question, however I attached a table of restriction enzymes from class and I was able to find AGCT and GATC in the sequence.
I'm not sure how to proceed.....

Answer 1

Hi, Just follow the following steps and you can be able to do the work.

• Open a new tab in the browser and type NEB cutter.
• Click on the NEB cutter link in the tab and paste your DNA segment in the prescribed area and click submit.
• The result will be loaded within a minute and that will contain all the list of Restriction enzymes that can cut the sequence.
• Tally the list with the Restriction Enzyme list of your class and those are actually the restriction enzymes needed for the work.

You can also provide the full list and the matched list of restriction enzymes for the work!

For the Primer designing follow the below steps-

• Open a new tab in the browser and type NCBI primer designing.
• Click on the NCBI primer Designing link in the tab and paste your DNA segment in the prescribed area and click "Get Primers".
• The result will be loaded within a minute and that will contain all the list of primers for this sequence.

However for manual primer designing which is actually complementary of the sequence form 5' and 3' you need to consider the following rules-

• Length of 18-24 bases
• 40-60% G/C content
• Start and end with 1-2 G/C pairs
• Melting temperature (Tm) of 50-60°C
• Primer pairs should have a Tm within 5°C of each other

• Primer pairs should not have complementary regions.