Tip collection VNTI exercises MBV4010 2015
Tip 1: How to import files from different formats into VNTI





In NCBI, identify the Genbank file for the protein (for instance NP_006011.2).
This file describes the protein - you want to find the CoDing Sequence (CDS), the region
of nucleotides that corresponds to the sequence of amino acids in the predicted protein.
The CDS includes start and stop codons, therefore coding sequences begin with an "ATG"
and end with a stop codon.
Identify the CDS link and click it.
Now you get the Genbank file for the CDS. Sometimes you will need to change the
settings under "Change region shown" to "Selected region" and Update View. (see below)
This file starts with a LOCUS ID and also contain a Gene ID (GI) . These numbers can be
used to identify the sequence.
LOCUS
DEFINITION
(ALKBH1),
ACCESSION
VERSION


CDS

NM_006020 1170 bp
mRNA
linear
PRI 20-JUN-2010
Homo sapiens alkB, alkylation repair homolog 1 (E. coli)
mRNA.
NM_006020 REGION: 10..1179
NM_006020.2 GI:87298839
The Genbank CDS file also contains information on the origin and history of the sequence
and publications connected to the sequence. Sometimes the Genbank file also contains
an ANNOTATION of the sequence, that is, it describes the location of exons, introns,
3`and 5`UTRs etc. Finally, it contains contains the CDS sequence and protein sequence.
In our case we can identify the the CDS and importantly, we see that its product is the
protein NP_006011.2 (see below).
1..1170
/gene="ALKBH1"
/gene_synonym="ABH; ABH1; alkB; ALKBH; hABH"
/EC_number="4.2.99.18"
/note="alkylation repair, alkB homolog; DNA lyase ABH1;
alpha-ketoglutarate-dependent dioxygenase ABH1"
/codon_start=1
/product="alkylated DNA repair protein alkB homolog 1"
/protein_id="NP_006011.2"
To make this sequence a VNTI file you have three options, described under A, B and C below:
1
A. The Genbank method. In the Genbank window in your browser: Select "Send".
You can now save the information in the Genbank file to the desktop. Save it as a Genbank file with the
extention".gb". Select file format "All files". (See below)
Open VNTI Explorer. In the upper menyu, select "DNA/RNA" (in some versions it is also called “Table”)
- "Import" - and "Molecule from text file".
Select the import format (Genbank).
Under GENERAL give the molecule a name.
Under DNA/RNA MOLECULE select if linear or circular. OK.
The sequence will appear in your database in VNTI Explorer.
Click the sequence file and it will open in Vector NTI. This file will now include all the information
availabile in the Genbank file, and the sequence is ANNOTATED.
B. The Gene ID method. In the Genbank file, identify the Gene ID (GI).
VERSION
NM_006020.2
GI:87298839
Open VNTI Explorer. In the upper menu, select "Tools" - "Open link" and "GID". Enter or paste the GID. OK. Select the import format
(Genbank).OK.
The Genbank file information will now be automatically downloaded
and opened in Vector NTI. Save the file in the VNTI Explorer Database.
2
C. (If nothing of the above works) The Copy/Paste method: Copy the CDS sequence. Open VNTI
Explorer. In the upper menyu, select "DNA/RNA" (in some versions it is also called “Table”) – then
select "New" - and "Molecule using sequence editor". Under GENERAL give the molecule a name.
Under DNA/RNA MOLECULE select if linear or circular. Under SEQUENCE AND MAPS - click "edit
sequence". Paste the sequence you copied previously and click OK. The sequence will appear in your
database in VNTI Explorer. Click the sequence file and it will open in Vector NTI
Tip 2:

You can use the import methods described under Tip1 b) and c). If you copy the DNA
sequence into VNTI, you must annotate the vecor using the information from your
compendia. If you import a “.gb” file the vector is already annotated. Please note that the
sequence download file is "zipped" and needs to be "unzipped" before you select it from
VNTI Explorer.
Tip 3:




You can copy, paste and delete sequences in Vector NTI the same way you do it in a text
document.
The functions in VNTI can help you to navigate in the sequence and find where to insert:
You can show the restriction sites in the vector polylinker by using the "Molecule
Display Setup" button in the menu.

In the dialog window select "Rmap Setup" then select the restriction endonucleases
indicated in your lab
manual (Fig2) and click
OK. The restriction sites
will now be shown on the
plasmid.

Click and select a region around the restriction sites. Then click the "View molecule
fragment " button. Now only the selected region will be displayed. Eventually, select an
even narrower region to display only the information relevant for you.
3

To translate a stretch of DNA, use the Translate button. Note that
you must indicate which strand is translated.

To mark a part of a sequence, mark like in a text document or in the graphics
panel, then click the "Add Feature" button and give it a name. In this way you
can for instance mark a region to be deleted and the immediate regions
surrounding it, the translational start site (ATG), the stop site, etc.

If you need to reverse a DNA fragment (make reverse
compliment) - select the molecule , then select the "Edit"
menu - "Molecule Operations" - then "Advanced
DNA/RNA", then "Reverse selection".
If you make a reverse compliment the annotation is lost (so
only do this if really required, and then do it with the
molecule that is the least annotated.

Tip 4:


You learned this in Tip 3.
Remember that also the vector backbone can be digested and will contribute to the
digestion pattern you observe.
Tip 5:
This is for pET-28-hABH1-del(348-389), pET-28-AlkB-L170A goes in a similar fashion












You can enter or import oligos (primers) into VNTI Explorer:
In the Explorer, select the oligos pane. This shows you a
database that contains your oligos.
Select the "New" button
Give your new oligo a name (like h1-fwd1) and
enter or copy the oligo sequence.
Now you can visualise your oligos in your
vector or molecule using the "Molecule Display
Setup" button in the menu (see Tip 3).
In the dialog window select "Motif" then select the primers you
want to visualise. (Since, in this exercise, the primers are not always matching 100%,
select the "Best fit" option.
Now you can see where your primers bind in the sequence.
Mark the fragment indicated by the primer-sites (remember to include the primers
themselves).
Copy the fragment and make a new molecule. Check that your primers bind in the
sequence.
Check where the restriction enzymes used in this exercises bind (if at all).
Identify the portion of the h1-Cdel-rev primer not complementary to the sequence; delete
this region and type in the new sequence that will be generated using these primers. You
know that you typed the right sequence if the identity of your primers now turned into
100%
You should see that you now have both restriction sites
Now you have created the PCR product used in the cloning. (Mark it using the "Add
features" button).
4



In the recipient vector identify the restriction sites used for cloning. Mark it using the "Add
features" button. Also mark a short stretch of bases immediately flanking the restriction
fragment. (This makes it easier to follow the next operations).
Now you have both the donor and recipient molecules and can delete the restriction
fragment in the recipient and insert the restriction fragment from the donor by copy paste.
Save the new molecule under its new name. Check that the new length of the vector is
correct. Translate the sequence to see that it is correct. (You can identify open reading
frames using the "Molecule Display Setup" and the option "ORFs".
Tip 12



Select the region to be amplified by RT-PCR (use the primers and the "Motif" function).
In the “Cloning” menu select "TOPO cloning" and Amplify selection".
Choose correct termini (T-A). Proceed to Cloning Wizard and Select the
recipient/destination vector.
Tip 14



Open the program Invitrogen/Contig Express and drag your sequencing file into the open
frame.
Double click the imported object and view your sequence and chromatograms.
Send your file to VNTI using the Project/Export…/Send to VNTI menu. Save the file in your
folder.
Tip 16




Select the full length reading frame that should be sub cloned into the donor vector.
In the menu "Cloning", select "Gateway Cloning" / "Create an entry clone" / "Amplify
selection to use in BP reaction".
Select the right strand for amplification.
Proceed to cloning wizard and save to database.
Tip17



In the menu "Cloning", select "Gateway
Cloning" / "Create an expression clone"
Select the donor and recipient vector
Proceed to cloning wizard and save to database.
5