In: Biology
Experiment 2: Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector
Bacteria frequently contain extrachromosomal plasmids, which are circular DNA genetic elements that are self-replicating. Plasmids are typically not necessary for the bacteria's survival but can sometimes confer a growth advantage to the bacteria. Plasmids can be transferred between bacteria and recombinant DNA technology makes use of this feature to introduce “foreign” genes into bacteria and use the bacteria as a cell “factory” to produce the gene. Genes are inserted into plasmids (also called vectors) by means of restriction enzymes. Restriction enzymes are bacterially derived enzymes that recognize specific DNA sequences and cut (or restrict) that particular DNA sequence in a consistent way. That is, any DNA that contains a particular restriction enzyme sequence will be cut the same. Restriction enzymes often produce overhanging ends of DNA (called sticky ends) that can adhere to each other through hydrogen bonding then DNA ligase permanently links the pieces together by forming covalent bonds between the phosphate backbones of the DNA, creating a recombinant or genetically engineered DNA. In this experiment, you will be given two sequences of DNA: one sequence is a foreign gene and the other sequence is of a plasmid vector. You will use a computer program to identify where the common restriction sites for both sequences occur. Scientists perform this type of computer simulation prior to performing restriction enzyme digestion to ensure that they will cut the sequences as expected.
Materials NEB Cutter Website:
http://tools.neb.com/NEBcutter2/index.php |
Procedure
Type the link listed in the materials box for the NEB Cutter website into a web browser (note, cutting and pasting the link from a PDF file format will not work. You must manually type in the address).
Copy and paste the foreign DNA sequence into the box on the NEB Cutter website where it says “Paste In Your DNA Sequence”. Foreign DNA Sequence:
GAATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT
CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC
GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG
CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGC
AGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG
CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCA
ACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA
TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC
TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC
GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC
TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCC
CGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGC
GCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG
GACGAGCTGTACAAGGGATCC
Title the sequence as “Foreign DNA” in the “Name of Sequence” box. Leave the other settings on their default status.
Click the “Submit” button (located to the right of the pasted DNA sequence).
On the new page that opens, select “Custom Digest” under the “Main Options” heading on the left side of the page.
The new page that opens will contain a list of restriction enzymes. Select the enzymes BamHI and EcoRI then click on the “Digest” button at the bottom of the page.
On the new page that opens you will see a linear representation of your DNA. Select “Fragments” under the “List” heading on the right side of the page.
Record the length of the longest fragment in Table 1. This fragment contains the sequence of the foreign DNA. The short fragments are the left over ends from the restriction enzyme digest. Close this window.
Return to the NEB Cutter homepage. Copy and paste the plasmid gene sequence into the box on the NEB Cutter website where it says “Paste In Your DNA Sequence”. Plasmid DNA Sequence:
GTTAACTACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG
TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA
AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCG
CCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACG
CTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATC
GAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT
TCTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTG
TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT
TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAG
AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT
CTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG
GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA
ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAAC
TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATG
GAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGG
TTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGC
ACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGT
CAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT
TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTACCCC
GGTTGATAATCAGAAAAGCCCCAAAAACAGGAAGATTGTATAAGCAAATATTTAAATT
GTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTT
AACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATA
GGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCC
AACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATC
ACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAA
AGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCGAACGTGGCGAGAAAGGAAG
GGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGC
TGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTAAAA
GGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT
TTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCC
TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGT
GGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG
CAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTT
CAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC
TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC
GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG
GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC
CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGA
ACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCC
TGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG
GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG
CTGGCCTTTTGCTCACATGTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTT
ACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC
ACAGGAAACAGCTATGACCATGATTACGCCAAGCTACGTAATACGACTCACTATAGGG
CAGATCTTCGAATGCATCGCGCGCACCGTACGTCTCGAGGAATTCCTGCAGGATATC
TGGATCCACGAAGCTTCCCATGGTGACGTCACCGGTTCTAGATACCTAGGTGAGCTC
TGGTACCCTCTAGTCAAGGCCTATAGTGAGTCGTATTACGGACTGGCCGTCGTTTTAC
AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCC
CCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACA
GTTGCGCAGCCTGAATGGCGAATGGCGCTTCGCTTGGTAATAAAGCCCGCTTCGGCG
GGCTTTTTTTT
Title this sequence as “Plasmid DNA” in the “Name of Sequence” box. Select “Circular” by the heading “The sequence is:”. Leave the other settings on their default status.
Click the “Submit” button (located to the right of the pasted DNA sequence).
On the new page that opens, select “Custom Digest” under the “Main Options” heading on the left side of the page.
The new page that opens will contain a list of restriction enzymes. Select the enzymes BamHI and EcoRI then click on the “Digest” button at the bottom of the page.
On the new page that opens you will see a circular representation of your DNA. Select “Fragments” under the “List” heading on the right side of the page.
Record the length of the longest fragment in Table 1. This fragment contains the majority of the plasmid DNA sequence. The short fragment is the excised plasmid DNA that lies in between the two restriction enzyme sites.
Table 1: Fragment Lengths | |
DNA Type | Longest Length (in base pairs) |
Foreign | |
Plasmid |
Q: Identify where the common restriction sites for both sequences occur?
The final results after completing all the steps are as below:
The restriction site after custom digest is shown below:
As we can see, in foreign DNA, ECoRI site is interfered by CpG island. So only BamHI is present in boh foreign DNA and plasmid DNA.
Identify where the common restriction sites for both sequences occur?
Common restriction sites occur by BamHI