Bioinformatics Tools Training Program

LAB
Free
  • 18 lessons
  • 0 quizzes
  • 4 week duration

Restriction Mapping & ORF Finder

The Type II restriction enzymes are among the most valuable tools available to researchers in
molecular biology. These enzymes recognize short DNA sequences (4–8 nucleotides) and cleave at,
or close to, their recognition sites (1,2). A comprehensive database (REBASE) contains information
about these enzymes including their recognition specificities and their sensitivity to DNA
methylation.

NEBcutter consists of a set of cooperating program modules. The module that finds recognition
sites implements a brute force algorithm in C (gcc version 2.96, http://www.gnu.org/software/gcc).
NEBcutter accepts an input sequence, which can be pasted in, picked up from a local file or
retrieved from NCBI as a GenBank file via its accession number. Various options are available to
select the size of ORFs to be displayed and the set of restriction enzymes to be used. The program
calculates the positions of all restriction enzyme sites noting those that might potentially be blocked
by overlapping methylation and finds the ORFs in the sequence. It then displays a schematic
diagram of the sequence, the long ORFs, based on the rules described in the Methods and all
restriction enzymes that cut it just once. The initial display also shows the enzymes that can be used
in a complete digest to excise each ORF that is displayed.

ORF Finder searches for open reading frames (ORFs) in the DNA sequence you enter. The program
returns the range of each ORF, along with its protein translation. Use ORF Finder to search newly
sequenced DNA for potential protein encoding segments. ORF Finder supports the entire IUPAC
alphabet and several genetic codes.
Procedure
Open the browser and go to the home page of ORF Finder. Figure 1 shows the ORF finder main
page at NCBI.

FASTA format is a simplest sequence format which starts with a ‘>’ symbol followed by the
sequence ID, other comments and computationally represented protein sequence.

Enter a computerized nucleotide sequence in the box provided (in FASTA format).

There is a drop down menu to select a genetic codon dictionary. It contains 20 different codon
dictionaries that contains codons for different organisms and organelles.Select any from the list
which you want for the search method.

In the genetic codes list, one can see many codon dictionaries. It differs in the triplets those codes
for amino acids.

Now press the ORF finder button to get the result .The result shows the all the possible six reading
frame present in the entered sequence query. One can see that the ORF is listed according
to their size and the graphical representation of the sequence.

Click on the green region which represent the ORF in the sequence, to see the ORF. Once you click,
it will turn into purple color indicating that the particular ORF is selected. The selected ORF is also
indicating in the list. It also displays the length and location of the selected ORF.

One can see the sequence of the selected ORF which actually codes for the protein. The user can
find the start codon, stop codon and the total number of the amino acids from the sequence. Now
click on Accept button.

The accepted region will turn into light green color indicates that the selected ORF is accepted. This
is to indicate that the user wants to study further about the particular ORF User can also perform a BLAST search for the particular ORF that we selected. Select the appropriate program and database. Then click on the BLAST button.