This document summarizes various bioinformatics analyses performed on the pyruvate kinase gene sequence from Vibrio cholerae, including restriction mapping, finding the open reading frame, multiple sequence alignment, phylogenetic tree construction, and primer design. Restriction mapping identified several restriction enzyme cutting sites. The open reading frame encoding pyruvate kinase was identified. A phylogenetic tree grouped pyruvate kinase sequences into two clades. Four primer pairs were designed for PCR amplification.

1. Department of Biochemistry and Molecular Biology
University of Dhaka
MS Practical
Bioinformatics Practical Note
Submitted by
Md. Atai rabby
MS
Biochemistry and Molecular Biology

2. Restriction Mapping
Tool : Restriction Mapper 3.0
Sequence : Pyruvate Kinase, Vibrio cholera O395
Name: Pyruvate Kinase
Conformation: linear
Overhang: five_prime, three_prime, blunt
Minimum Site Length: 4 bases
Maximum Number of Cuts: all
Included: all commercial, prototypes only
Noncutters: AarI, AatII, AbsI, AclI, AcyI, AflII, AjuI, AlfI, AloI, AlwNI, ApaI, AscI, AsuII, AvaI, BaeI,
BamHI, BarI, BbvCI, BdaI, BglI, BglII, BplI, Bpu10I, BsaXI, BsePI, BseRI, BsmI, Bsp1407I, BspHI,
BsrBI, BsrDI, BstEII, BstXI, BtgZI, ClaI, CspCI, DraII, DraIII, DrdI, Eam1105I, EciI, Eco31I, EcoNI,
EcoRI, EcoRV, FalI, FseI, FspAI, GsuI, HaeIV, Hin4I, HindIII, KpnI, MauBI, MfeI, MluI, NaeI, NarI,
NcoI, NdeI, NheI, NlaIV, NmeAIII, NotI, NruI, NspI, PacI, PasI, PflMI, PfoI, PmeI, PpiI, PpuMI, PsiI, PI-
PspI, PspXI, PsrI, PstI, PvuI, PvuII, RsrII, SacI, SacII, SalI, SanDI, SapI, PI-SceI, SexAI, SfiI, SgfI, SgrAI,
SgrDI, SmaI, SpeI, SphI, SrfI, Sse8387I, SspI, StuI, SwaI, TaqII, TauI, TstI, Tth111I, VspI, XbaI, XcmI,
XhoI, XmnI
Name Sequence
Site
Length
Overhang Frequency Cut Positions
BalI TGGCCA 6 blunt 1 263
Eco47III AGCGCT 6 blunt 1 506
SnaBI TACGTA 6 blunt 1 1057
AflIII ACRYGT 6 five_prime 1 1401
Hpy99I CGWCG 5 three_prime 3 382, 834, 892
MboII GAAGA 5 three_prime 3 762, 772, 987
ArsI GACNNNNNNTTYG 7 three_prime 4 28, 60, 520, 552
NlaIII CATG 4 three_prime 4 84, 723, 909, 984
TspGWI ACGGA 5 three_prime 4 139, 205, 571, 598
AluI AGCT 4 blunt 5 66, 228, 622, 705, 1219
AciI CCGC 4 five_prime 5 632, 883, 970, 1094, 1138
TspRI CASTG 5 three_prime 7 417, 423, 1068, 1121, 1281, 1370,

3. Name Sequence
Site
Length
Overhang Frequency Cut Positions
1395
FaiI YATR 4 blunt 8 21, 82, 91, 388, 721, 907, 982, 1074
GlaI GCGC 4 blunt 8
313, 450, 506, 876, 986, 1112, 1310,
1362
MaeII ACGT 4 five_prime 10
119, 353, 596, 729, 810, 864, 890,
1050, 1056, 1403
CviJI RGCY 4 blunt 12
32, 66, 228, 263, 622, 677, 705,
1046, 1069, 1180, 1219, 1322
SetI ASST 4 three_prime 28
68, 122, 207, 230, 350, 356, 471,
479, 533, 591, 599, 624, 707, 732,
740, 744, 813, 867, 893, 954, 1053,
1059, 1083, 1221, 1242, 1295, 1299,
1406
Discussion
From the analysis we can come to following analysis
1. There are many cutting sites for our sequence by commercially available Restriction enzyme
2. Some enzymes do not have any cutting site in the sequence
3. Many of the enzymes here are frequent cutter(4 cutter)
4. SetI have highest cutting sites in this sequence
5. BalI, Sna BI is not suitable for plasmis construction as they produce blunt overhang.

4. Finding Open Reading Frame
Tool : ORF finder
pyruvate kinase : Vibrio cholerae O395
Discussion
NCBI ORF finder produce six possible reading frames, we cheched each reading frames and blast
them with nr nucleotide database which ultimately proof that the first ORF with length 1413 is the
best match ORF

5. Multiple Sequence Alignment
Tool : Clustalw
Algorithm : Clustal W
Discussion
ClustalW alignment could be now use to find conseve regions, bulding phylogenetic analysis,
calculating conservancy or divergency, finding domains etc.

6. Making Phylogenetic tree from a Multiple Alignment
Tool : clustalW
Method: Neighbour Joining Method
Discussion
We used four strain of vibrio cholerae and their two type of Pyruvate Kinase Product in this
analysis.
The following decision could be made from the tree
1. The tree is devided into three clades
2. First clade contains only Pyruvate kinase II enzyme group of four Vibrio strain
3. The third clade contain only Pyruvate kinase enzyme group of four Vibrio strain
4. Pyruvate Kinase and Pyruvate kinase II evolve differently in vibrio cholerae srtarin

7. Primer Designing
Tool : Primer 3, IDT Olig Analyzer
Found Primer 1 LEFT PRIMER : GGTAACAAAGAGCGCGTAGC
RIGHT PRIMER: CGCTTCAGTTGCTACGATGA
PRODUCT SIZE: 84
2 LEFT PRIMER : CGGTGACTACGTTGAACACG
RIGHT PRIMER: GTCGCCGTGATAACCACTTT
PRODUCT SIZE: 723
3 LEFT PRIMER : ATGGAAGTACTGGCGACCAC
RIGHT PRIMER: TTCTTGTTGGTGGTGACTGC
PRODUCT SIZE: 818
4 LEFT PRIMER : TTGAACACGGCACTCGTATC
RIGHT PRIMER: GTCGCCGTGATAACCACTTT
PRODUCT SIZE: 712
Features of First Primer LENGTH:20
GC CONTENT:55.0
%MELT TEMP:56.4 ºC
MOLECULAR
WEIGHT:6200.1 g/mole
EXTINCTION COEFFICIENT:205600L/(mole·cm)
nmole/OD260:4.86
µg/OD260:30.16
Hairpin Ananlysis
[ Value of ΔG and Hairpin indicating
the primer as poor candidate ]
ΔG (kcal.mole-1
) : -1.16
Hetero dimer
[Value of ΔG indicating Low
possibility of Hetero dimer formation ]
ΔG = -8.35 kcal/mole, -6.75 kcal/mole, -4.74 kcal/mole