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Question: Part B – Using BLAST to identify SNPs compared to a reference sequence BLAST (Basic Local Alignme…

by | Dec 5, 2023 | Posted Questions

Part B – Using BLAST to identify SNPs compared
to a reference sequence

BLAST (Basic Local Alignment Search Tool) is often used to
compare unknown nucleotide or amino acid sequences to sequences in
online databases. It also can be used to compare variant sequences
to a standard “reference” sequence that is entered manually.

Your research group has now sequenced the entire coding region of
the insulin cDNA (330 nucleotides) from the five SNP variants in
Part A. These sequences are shown below, along with the wild-type
reference sequence.

Reference insulin cDNA sequence (wild-type)
ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGAC
CCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTAC
CTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGAC
CTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTG
GCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGC
TCCCTCTACCAGCTGGAGAACTACTGCAAC

Patient 1 sequence
ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGAC
CCAGCCACAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTAC
CTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGAC
CTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTG
GCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGC
TCCCTCTACCAGCTGGAGAACTACTGCAAC

Patient 2 sequence
ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGAC
CCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTAC
CTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGAC
CTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTG
GCCCTGGAGGGGTCCCTGCAGAAGTGTGGCATTGTGGAACAATGCTGTACCAGCATCTGC
TCCCTCTACCAGCTGGAGAACTACTGCAAC

Patient 3 sequence
ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGAGGACCTGAC
CCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTAC
CTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGAC
CTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTG
GCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGC
TCCCTCTACCAGCTGGAGAACTACTGCAAC

Patient 4 sequence
ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGAC
CCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTAC
CTAGTGTGCGGGGCACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGAC
CTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTG
GCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGC
TCCCTCTACCAGCTGGAGAACTACTGCAAC

Patient 5 sequence
ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGAC
CCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTAC
CTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGAC
CTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTG
GCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTGCCAGCATCTGC
TCCCTCTACCAGCTGGAGAACTACTGCAAC

You decide to use BLAST to quickly identify the nucleotide changes
in the five SNP variants compared to the wild-type reference
sequence. Go to the BLAST website by clicking the Launch
BLAST button. Then follow the instructions below.

Question: Part B - Using BLAST to identify SNPs comparedto a reference sequenceBLAST (Basic Local Alignme...

BLAST Search Instructions

In the middle of the page, under the Basic
BLAST heading, click nucleotide blast. A
new page will appear.

In the Enter Query Sequence panel, check the
box next to Align two or more sequences, and wait
for the page to refresh.

Copy the reference sequence shown above, and paste it into the
Enter Query Sequence box at the top of the
page.

Copy the sequence for patient 1, and paste it into the
Enter Subject Sequence box.

In the Program Selection panel, select
Highly similar sequences (megablast).

Click the BLAST button near the lower-left
corner of the page. Wait for BLAST to complete the search, which
may take 10-30 seconds.

A new page displays your results. (If your search returns no
results, see Hint 1 to determine what went wrong.) Scroll down to
the Alignments section to see the two sequences aligned. The
reference sequence will be on top (Query 1), and the patient 1
sequence will be below it (Sbjct 1). The screen should look similar
to this example:Question: Part B - Using BLAST to identify SNPs comparedto a reference sequenceBLAST (Basic Local Alignme...

Note that identical nucleotides are indicated with a line drawn
between them. Scan the alignment to find any nucleotides that are
not identical, and write down the nucleotide number and the patient
and reference nucleotides at that number. In the example shown
above, nucleotide 24 is an “A” in the patient sequence and a “G” in
the reference sequence.

Return to the BLAST page by clicking the Edit and
Resubmit link in the upper left. Clear the Enter
Subject Sequence box by clicking Clear
above the box. Then repeat steps 4-8 for the other four patients.
(You may leave the Enter Query Sequence box
unchanged, as it will still contain the reference sequence.)

Using the results of your BLAST search, complete the table below
by dragging one blue label to each blue target and one pink label
to each pink target. Labels may be used once, more than once, or
not at all.

NOTE: Be sure to enter the patient’s nucleotide
information in the middle column and the
wild-type nucleotide information in the
right-hand column.

Question: Part B - Using BLAST to identify SNPs comparedto a reference sequenceBLAST (Basic Local Alignme...

Launch BLAST

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