Download HaeIII restriction endonuclease was used to digest the following

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Cell Biology, FA 02. This problem set is worth 25 points and counts as two problem set grades, PS 3 and
PS 4. Due Thursday, 12-12 at 9 am but use this as part of your review for the final exam. Answer these
questions on separate sheets of paper and attach it to the exam
DraI restriction endonuclease was used to digest the following DNA piece from the globin gene. Note
that only the 5’ to 3’ non-template sequences are shown. The 3' to 5' template sequence would be
3'taaatccaat…. Etc.
5’ATTTAGGTTATTTGCAGAAAACTGAGGCCTAATGGTAATAGCTTTTTAAATTAATTTATTTT
TAATACAAGGAGAACAGATTTAGATGTAGTTCATACATACAATTTTAAGAATGTAATGATGG
AGAGAGGCAGGGAGAAGGATAATGGTTTTTATTTTCTGGACTTGTACAACTCTGTATCTTAT
TCACTGTAACAGTATTTTTCAGGGCAGTAACCACAAAAAAATGTGAATTTACAAAAACTCAA
CAAACCTGTTGTGAGTTAATCTATTGTAATTGTGAAGGTAAAAGGCACACTTCCCCAGTGAA
TCACTTAAGCAGGAAAAACTGGACACATTTGAAAGAATAAATTCCTTGACTATTCTGCAAAG
ATAAGCGCAGGGACCTGGGAAGAGGGGTATTGTCCTCAACTGGTATGAGGATTTGACTCAG
ACAGCATCAAGTACAATTGCCTTGTGTTTACTTTTAGAATAAGAGAGTTTTGAGTCCCATAAT
CAAAAGAGGAGACCCAGGGTGCACATAGATCTCTGAATGCCTTCAGTGTGAGAAGAGATAT
GACCTGAGTTCATCAGCCATAAGCCCTTTCCCCTCTCTCCCACTCAGCAGCCAGGTCAGAGT
GAGCTTGATCACCCTGCCCCTGTGCTGGTTTGACTCACTCCTAGGCCCAGCCATGGAAGGAG
GTCCTGTCTGGGCTCAGACAGCACTATCTGCAGGGCCAGGACTTAGGCGGGGCTGGTAGGCT
CTAGAAAGTTTCCCTCCCAGCAAGTGAGGAAGGTTGGCTTATTTACATATTTAAGTGTCATA
TGTATTTTTAAAGAAATAACTAGTAGCCTGCCCAACTTGGGCATAGACAGCACCTAGAAAAT
TTTACAATCCTTCTGTTCTGTCTCTCACAGGAGCACAATTAGAGGCAAAGGGCACTCTCGAA
AGTGCCTGAGTCAGAGCCTG- 3’
1. (4 pts) You want to digest this DNA with the restriction enzyme, DraI. Your stock DNA
concentration is 1 g/L; your stock DraI enzyme concentration is 1 unit/L, your enzyme stock
buffer (50 mM Tris buffer, pH 8.00; 50 mM MgCl2; 0.5 M NaCl; 50 mM Dithiothreitol) is 10X
concentration. You want to make a final reaction mixture, to be incubated at 37oC, containing the
following in a total volume of 100 L: 5 g of DNA, 10 units of restriction enzyme DraI and 1 X
buffer.
A. Give the volume of each stock component and distilled water that would be added to give the
final amounts and concentrations in the 100 L of your final reaction mixture.
B.
2.
What are the final concentrations, in ?g/mL of DNA; ?units/mL of restriction enzyme DraI;
?mM Tris buffer; ?mM MgCl2; and ?mM Dithiothreitol, of these components in your final
reaction mixture.
(4 pts) Using any of my catalogues,
A. what is the DNA palindrome that DraI cuts the phosphodiester bonds at and where does it cut
within the palindrome?
B. Does it give blunt ends or overhangs (“sticky ends”) ends in the pieces of DNA produced?
C. This enzyme originally came from what organism (genus and species)?
D. What is the purpose of this enzyme in this organism?
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3. (2 pts) Circle the sites of cleavage for DraI enzyme in the partial DNA single strand gene above and
label the fragments with numbers or letters. (Hint: Use the “find” function under the edit drop down
menu and search for the 5’ – 3’ nucleotide palindrome sequence until you find all of the sites. I will
post this problem set on the web or send it to each of you by email.)
4.
(1.5 pt) Determine the sizes, in base pairs (bp) and kilobase pairs (kb), and mega base pairs (Mb) of
each of the DNA double stranded pieces generated by the enzyme. Label each fragment with
numbers or letters and give their sizes. (Hint: use the “word count” function under the tools drop
down menu and count the number of letters from whatever to whatever. I will post this problem set
on the web or send it to each of you by email.)
5.
(2 pt) On a 1% agarose gel, draw, roughly, a representative gel with wells on a separate sheet of
paper, illustrating the relative mobility of your labeled fragments from 3 and 4.
6. (2 pt) On a 2% agarose gel, draw, roughly, a representative gel wells on a separate sheet of paper,
illustrating the relative mobility of your labeled fragments from 4. How does the mobility of the
fragments here compare to the mobility of the fragments in 5?
7. (2 pt) Find any one start codon in a mRNA that could come from the above non-template sequence
and give me the nucleotide sequence of the first ten (10) codons beginning with the first start codon in
the partial mRNA sequence. (Hint: Find any ATG in the non-template strand: remember that,
except for the T to U change for the RNA, it is the identical sequence as in the non-template strand!).
Correctly label your mRNA, 5' end and 3' end.
8. (2 pt) Give me the partial amino acid sequence, 3 letter code and 1 letter code, of the polypeptide
containing the ten (10) amino acids that would be specified by the mRNA that you put in 7. Correctly
label your peptide, amino end to carboxyl end.
9.
(2 pt) Give an additional different mRNA sequence that would code for the same amino acids in 8.
Correctly label your mRNA, 5' end and 3' end.
10. (up to 1.5 pts, 0.5 pts each) Assuming that this is a eukaryotic mRNA:
A. What is one other non-translated RNa sequences that would be present in the primary mRNA as it
is first transcribed from the template strand of the DNA?
B. What are 2 other non-translated RNA sequences, added after transcription (post- transcriptional)
in the processed mRNA that is found in the cytoplasm?
11.
(2 pts). Determine whether the restriction enzyme, SmaI, cuts this DNA fragment using the same
procedure that you used in #3. Give its palindrome sequence and where it cuts within this sequence
and whether it gives overhang or sticky ends or blunt ends.
12.
Bonus: (2 pts). Determine whether the restriction enzyme, CfoI, cuts this DNA fragment using the
same procedure that you used in #3. Give its palindrome sequence and where it cuts within this
sequence and whether it gives overhang or sticky ends or blunt ends. Give the sizes in bp of the
pieces of DNA that results from cutting with this enzyme.
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