Question

Below is a DNA sequence of Borrelia burgdorferi containing the sequence of one strand only.

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3’

A) Please make the DNA double stranded by writing the complementary strand of the DNA underneath the strand above.

B) Find in internet restriction sites for BamHI, EcoRI and XbaI. Please place a box around each restriction site in the DNA.

C) You choose to cut this DNA with all three restriction enzymes.  How many DNA fragments will you get?

D) You choose to cut this DNA with BamHI and XbaI, please diagram the products you will get.

Answer 1

QA) Complementary sequence:

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3’
3'-TGAAGTCCTAGGTGACCCGGGCTTAAGCAGGACTCGAGATCTCAGGAAGC-5'

Accordi to chargaff's rule A always pair with T and G always pairs with C and vice versa.

QB) Restriction site:

Restriction sites are the recognition sites which are located on a DNA molecule which are recognized by restriction enzymes.

BamHI :

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3’
3'-TGAAGTCCTAGGTGACCCGGGCTTAAGCAGGACTCGAGATCTCAGGAAGC-5'

EcoRI :

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3’
3'-TGAAGTCCTAGGTGACCCGGGCTTAAGCAGGACTCGAGATCTCAGGAAGC-5'

XbaI:

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3’
3'-TGAAGTCCTAGGTGACCCGGGCTTAAGCAGGACTCGAGATCTCAGGAAGC-5'

QC) Generally restriction enzymes cleaves restriction site at different position and develops sticky end fragments or blunt end fragments.

BamHI cleaves after the first G and develops 2 separate fragments with sticky ends.Likewise ECoRI cleaves after the first G and develops 2 separate fragments with sticky ends.Likewise XbaI cleaves after the First T and develops 2 fragments.
So in total 6 restriction fragments were developed for the above sequence with the help of the three restriction enzymes.

BamHI :

Fragment 1    Fragment 2

5’-ACTTCAG 3'    5' GATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3’

3'-TGAAGTCCTAG 5'       3' GTGACCCGGGCTTAAGCAGGACTCGAGATCTCAGGAAGC-5'

EcoRI:

5’-ACTTCAGGATCCACTGGGCCCG 3'   5' AATTCGTCCTGAGCTCTAGAGTCCTTCG-3’
3'-TGAAGTCCTAGGTGACCCGGGCTTAA 5' 3' GCAGGACTCGAGATCTCAGGAAGC-5'

XbaI:

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCT 3' 5' CTAGAGTCCTTCG-3’   
3'-TGAAGTCCTAGGTGACCCGGGCTTAAGCAGGACTCGAGATC 5' 3'   TCAGGAAGC-5'

Q4) Products of DNA fragment when treated with BamHI and XbaI:

Generally the DNA sequence is taken to which the appropriate restriction enzymes were added which can recognise the specific site and digest the DNA in to fragments.Below diagram shows the product formation of DNA by treating with BamHI and XbaI.

BamHI :

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3’
3'-TGAAGTCCTAGGTGACCCGGGCTTAAGCAGGACTCGAGATCTCAGGAAGC-5'

  
End product:

5’-ACTTCAG 3' 5' GATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3‘

3'-TGAAGTCCTAG 5' 3' GTGACCCGGGCTTAAGCAGGACTCGAGATCTCAGGAAGC-5'

XbaI:

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCTCTAGAGTCCTTCG-3’
3'-TGAAGTCCTAGGTGACCCGGGCTTAAGCAGGACTCGAGATCTCAGGAAGC-5'

End product:

5’-ACTTCAGGATCCACTGGGCCCGAATTCGTCCTGAGCT 3' 5' CTAGAGTCCTTCG-3’
3'-TGAAGTCCTAGGTGACCCGGGCTTAAGCAGGACTCGAGATC 5' 3' TCAGGAAGC-5'

Hope this helps you to resolve your doubts:)