Homework 4 ans key. DO NOT COPY
FCH 532 Homework 4
This assignment is due Monday in class!
Put your name and student ID number on the top of each page.
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1. (a) Using the PAM-250 log odds substitution matrix and the Needleman-Wunsch
algorithm, find the best alignment of the two peptides PQRSTV and PDLRSCPSV.
P
Q
R
S
T
V
P
6
0
0
1
0
-1
D
-1
2
-1
0
0
2
L
-3
-2
-3
-3
-2
2
R
0
1
6
0
-1
-2
S
1
-1
0
2
1
-1
C
-3
-5
-4
0
-2
-2
P
6
0
0
1
0
-1
S
1
-1
0
2
1
-1
V
-1
-2
-2
-1
0
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First, transform the matrix and then determine the best alignment
P
D
L
R
S
C
P
S
V
P
21
12
13
7
7
3
12
5
-1
Q
13
15
11
7
5
1
6
3
-2
R
7
6
4
13
6
2
6
4
-2
S
6
5
2
5
7
5
6
6
-1
T
4
4
2
3
5
2
4
5
0
V
-1
2
2
-2
-1
-2
-1
-1
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Alignment:
PQ-RS-TV
PDLRSCPV
(b) What is its alignment score using a gap penalty of -8 for opening a gap and -2 for
every “residue” in the gap?
Alignment score = 21 + (-8-2*1) + (-8-2*1) = -1
(c) What is its normalized alignment score (NAS) using the scoring system of 10 for each
identity, 20 for each identity involving Cys, and -25 for every gap?
Using the scoring of 10 for each identity and 20 for Cys, -25 for the gap.
AS = 10 * 4 +20*0 -25*2 = -10
NAS=AS/length of shortest peptide*100= -10/6*100= -167
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Homework 4 ans key. DO NOT COPY
(d) Is this NAS indicative of a homology? Explain.
No these peptides are not homologous based on the NAS and peptide length. The
score is negative, which gives no chance for homology.
2. You have been given an unknown protein to identify. You digest it with trypsin and,
by using Edman degradation, find that one of the resulting peptide fragments has the
sequence GIIWGEDTLMEYLENPK.
Using BLAST, find the most probably identity (or identities) of the unknown protein.
Most likely cytochrome c
3. You have manufactured a DNA chip consisting of 4 rows and 10 columns in which the
Mth column contains DNAs of the sequence 5’-GACCTGACGT-3’ but with a different
base in the Mth position for each of the 4 rows (from top to bottom, G, A, T, and C).
Draw the appearance of the chip after it is hybridized with fluorescently labeled RNA of
sequence (a) 5’-ACGUCAGGUC-3’ and (b) 5’-ACGUCUGGUC-3’
5’
G
A
T
C
2
G
a
A
C
C
a
T
b
a
a
a
G
a
A
C
G
a
T-3’
a
a
a
Homework 4 ans key. DO NOT COPY
4. Describe in detail dideoxy sequencing (Sanger Method).
4 reactions are prepared with deoxynucleotides corresponding to each of the 4 bases and
with 1 each of the dideoxynucleotide. The dideoxynucleotide when incorporated into the
DNA sequence cannot be further extended because it lacks the 3’-OH group. This
terminates the reaction at that particular base. The products of DNA synthesis are
separated by gel electrophoresis based on their size and can be used (if labeled) to
determine the sequence.
5. Outline chemical synthesis of a chemical oligonucleotide.
See next page
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Homework 4 ans key. DO NOT COPY
1. Dimethoxytrityl (DMTr) protecting group at the 5’ end is removed with
trichloroacetic acid (Cl3CCOOH)
2. The 5’ end of the oligo is coupled to the 3’ phosphoramidite derivative. Tetrazole
is used as coupling agent.
3. Any unreacted 5’ end group is capped by acetylation to block its extension.
4. The phosphite triester group from the coupling step is oxidized with I2 to the
phosphotriester (more stable).
5. Treated with NH4OH to remove blocking groups and to remove from support.
6. Purified and separated by HPLC or gel electrophoresis
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