Suppose you have a PCR amplicon consisting of the sequence below. The primers used to generate the amplicon are: 5'AT3' and 5'GA3'. Which of the following is true?
a. If you use the 5'AT3' primer for a subsequent sequencing reaction, the sequence of the shortest fragment containing a chain terminating nucleotide is: 5'ATC3'
b. If you use the 5'AT3' primer for a subsequent sequencing reaction, the sequence of the shortest fragment containing a chain terminating nucleotide is: 3'TAG5'
c. If you use the 5'AT3' primer for a subsequent sequencing reaction, the sequence of the shortest fragment containing a chain terminating nucleotide is: 5'C3'
d. If you use the 5'AT3' primer for a subsequent sequencing reaction, the sequence of the shortest fragment containing a chain terminating nucleotide is: 5'G3'
Homework Answers
In DNA sequencing, sequence of any strand of DNA can be read.
The primer used should bind to the 5’ to 3’ strand of the DNA sequence. It is not binding to 3’ to 5’ strand as DNA polymerase can add nucleotides only at the 3’ end. Hence, the primer is actually read in 3’ to 5’ direction.
The Sanger sequencing fragments are shown in the image. Wherever there is a dideoxynucleotides added, there will be chain termination seen as ddNTPs lack the 3’ OH group required by DNA polymerase to add nucleotides. The shortest fragment will be the one in which ddCTP is added. This is because C is the next nucleotide to be added after the primer by DNA polymerase. The primer sequence will always be seen in the fragments produced as primers are used by DNA polymerase to add nucleotides. They are not cleaved off after the reaction. Hence, 5’C3’ or 5’G3’ cannot be the fragment seen.
The sequence cannot be 3'TAG5' as primer is not binding to the 3’ to 5’ strand as DNA polymerase cannot add nucleotides to 5' end. The ddNTP has to be added after T and not after A as the 3' nucleotide of the primer is T (5'AT3')
Right answer:
a. If you use the 5'AT3' primer for a subsequent sequencing reaction, the sequence of the shortest fragment containing a chain terminating nucleotide is: 5'ATC3'.
Add Answer to:
Suppose you have a PCR amplicon consisting of the sequence
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You want to generate billions of copies of a DNA fragment
(sequence) consisting of ONLY the bracketed sequence in the DNA
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bases long, let’s suppose for this example that 3 base long primers
will work successfully. Which of the following primers will work to
generate your PCR amplicon (DNA fragment)?
5'-AATGAGCCATC[CCATATAAGCCGCGAGTTTG CATGTAC---3' O 5'CCA3' alone O 5'CCA3' and 5'CAA3' O 5'TGG3' and 5'TTG3' O S'ATC3' and 5'TAC3' O...
1.) PCR a.) Using the dsDNA sequence 1 as a template, you want to generate dsDNA...
1.) PCR
a.) Using the dsDNA sequence 1 as a template, you want to
generate dsDNA sequence 2 using PCR. What
are the sequences of the two primers you use? 15nt of each
primer have to anneal perfectly to the template.
Write the primer sequences in 5’->3’ direction in the
provided space.
(25 points)
Sequence 1
5-TATAGGACGATGTTGATGAATGGTACAATCACAGTACGTACGTACAGTCAGTGAAA-3
Sequence 2
5-TAGCGGTACATGTTGATGAATGGTACAATCACAGTACGTACGTACAGTCTATCGAT-3
Primer 1 5-
-3
Primer 2 5-
-3
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please i need help with a, b, c this is the sequence 5’ATGTATTATTATTTTTTTGTTTTTTTTGCAATATATGCTAATGGATTGCTAAGAAATA AAGATCCTAACATTTTTGCGAG TAGCAATGATGAGATCATAGAAAATGATAAAAGTATGAATACCTTTGTTATGTCAAC AAATGGAAGTTTATATTTAAATA...
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this is the sequence
5’ATGTATTATTATTTTTTTGTTTTTTTTGCAATATATGCTAATGGATTGCTAAGAAATA
AAGATCCTAACATTTTTGCGAG
TAGCAATGATGAGATCATAGAAAATGATAAAAGTATGAATACCTTTGTTATGTCAAC
AAATGGAAGTTTATATTTAAATA
GTGATTTTAATTTAAATGAAGCATCCAACGAAAGCTTCTTAGAAAATTGCAATATCA
ATAGTTGTGTAGATATAGGTCAT
GAAAATGGCAACAAAATAAATAGTCAAGAAAATGAGCATGCTAAAAATAATAACA
ACAGTAATAATAACAATTTAAAACC
AGAATACAATAATAATAATAATAATTTAAAACCAGAATACAATAATAATAATTTAA
AACCAGAGTACAATAATAACAATT-3’
1. Polymerase chain reaction 5'- ATGTATTATTATTTTTTTGTTTTTTTTGCAATATATGCTAATGGATTGCTAAGAAATA AAGATCCTAACATTTTTGCGAG TAGCAATGATGAGATCATAGAAAATGATAAAAGTATGAATACCTTTGTTATGTCAAC AAATGGAAGTTTATATTTAAATA GTGATTTTAATTTAAATGAAGCATCCAACGAAAGCTTCTTAGAAAATTGCAATATCA ATAGTTGTGTAGATATAGGTCAT GAAAATGGCAACAAAATAAATAGTCAAGAAAATGAGCATGCTAAAAATAATAACA ACAGTAATAATAACAATTTAAAACC AGAATACAATAATAATAATAATAATTTAAAACCAGAATACAATAATAATAATTTAA AACCAGAGTACAATAATAACAATT-3' a) One strand of a chromosomal DNA sequence is shown above. How would you amplify and isolate a DNA fragment defined by the sequence shown in red, using polymerase chain reaction. Design PCR primers (Forward and Reverse primers, each 20 nucleotides long, that...
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1.) PCR
a.) Using the dsDNA sequence 1 as a template, you want to
generate dsDNA sequence 2 using PCR. What
are the sequences of the two primers you use? 15nt of each
primer have to anneal perfectly to the template.
Write the primer sequences in 5’->3’ direction in the
provided space.
(25 points)
Sequence 1
5-TATAGGACGATGTTGATGAATGGTACAATCACAGTACGTACGTACAGTCAGTGAAA-3
Sequence 2
5-TAGCGGTACATGTTGATGAATGGTACAATCACAGTACGTACGTACAGTCTATCGAT-3
Primer 1 5-
-3
Primer 2 5-
-3
1.) PCR a.) Using the dsDNA sequence 1 as a template, you...
please i need help with a, b, c
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5’ATGTATTATTATTTTTTTGTTTTTTTTGCAATATATGCTAATGGATTGCTAAGAAATA
AAGATCCTAACATTTTTGCGAG
TAGCAATGATGAGATCATAGAAAATGATAAAAGTATGAATACCTTTGTTATGTCAAC
AAATGGAAGTTTATATTTAAATA
GTGATTTTAATTTAAATGAAGCATCCAACGAAAGCTTCTTAGAAAATTGCAATATCA
ATAGTTGTGTAGATATAGGTCAT
GAAAATGGCAACAAAATAAATAGTCAAGAAAATGAGCATGCTAAAAATAATAACA
ACAGTAATAATAACAATTTAAAACC
AGAATACAATAATAATAATAATAATTTAAAACCAGAATACAATAATAATAATTTAA
AACCAGAGTACAATAATAACAATT-3’
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