CHAPTER 7
Detection of Restriction Enzyme Sites

CS Mukhopadhyay and RK Choudhary

School of Animal Biotechnology, GADVASU, Ludhiana

7.1 INTRODUCTION

The restriction enzyme (RE) sites present on a nucleotide sequence can be detected using a suitable in silico tool. A nucleotide sequence is subjected to detection of RE site(s) in some wet‐lab experiments such as gene cloning, nucleotide sequencing, in vitro expression of a target protein, RFLP, AFLP, restriction mapping and restriction enzyme assays.

Several online tools for RE site detection are available. Some of the user‐friendly and accessible web‐based tools and their URLs (websites) have been tabulated below. Detailed procedures for determining RE sites using all these web tools will not be covered. This chapter will show how to use NEBCutter (New England Biolabs) as a tool for identifying RE sites.

  1. NEBCutter (http://tools.neb.com/NEBcutter2/index): New England Biolabs hosts this RE site mapper software.
  2. Webcutter 2.0 (http://rna.lundberg.gu.se/cutter2/): Another RE site detection software (online, free) for linear and circular DNA.
  3. Mapper (http://arbl.cvmbs.colostate.edu/molkit/mapper/index.html): Java platform‐based online software for mapping the RE sites on a target sequence.
  4. Web Map (https://pga.mgh.harvard.edu/web_apps/web_map/start): This tool maps the RE sites for circular or linear nucleotide sequences. The reverse complement of the given sequence can also be checked for mapping of RE sites.
  5. Restriction‐Mapper (http://restrictionmapper.org/): Online, freely available tool for mapping restriction endonuclease sites on a DNA sequence.

7.2 OBJECTIVE

To identify the RE site(s) present in a given nucleotide sequence.

7.3 PROCEDURE (USING NEBCUTTER)

7.3.1 Select the nucleotide sequence

This could be a nucleotide sequence of any length, depending on the purpose. Insert for gene cloning, or cDNA expression, an amplicon for detection of mutation (RFLP), or a DNA sequence which is to be screened for the presence of RE sites (RE assay).

7.3.2 Open the online tool for RE site detection, NEBCutter v2.0

This is hosted by New England BioLabs (Vincze et al., 2003); the URL is http://tools.neb.com/NEBcutter2/index.

7.3.3 Paste or upload the sequence

There are several options to enter the sequence (one at a time) under study:

  1. Local sequence file: Prepare a notepad (*.txt) file and maintain individual the DNA sequence in FASTA format. The maximum size of the file can be 1 Mb.
  2. GenBank Accession Number: Paste/type the NCBI, GenBank accession number in the text box provided. You can browse GenBank online to select or view the input sequence flat file.
  3. Paste the DNA sequence: You can directly paste the input DNA sequence (in FASTA format) in the sequence box provided. The maximum input limit is 300 kilobases.
  4. Standard sequences‐ # Plasmid vectors: This is a drop‐down menu from which you need to select your plasmid vector for RE analysis. Keep the parameters (e.g., ‘Local sequence file’, ‘GenBank Accession Number’ and ‘Paste in your DNA sequence’) blank to screen the Plasmid vector.
  5. Standard sequences‐ # Viral + phage: Similar to the above, but enlist the sequences of phage and viruses.

7.3.4 Selection of options

  • Linear or circular: Select the appropriate radio button to indicate whether your input sequence is a linear or circular sequence.
  • Enzymes to use: There are five alternatives, of which you can select any one. The first three options enable you to select enzymes from New England Biolabs (NEB) or other commercially available ones. The last two options allow you to choose some defined oligonucleotide sequence(s). There is a link “[define oligos]” which allows the user to define his/her own oligos.
Display window of nc2.neb.com/NEBcutter2/index_oligos.php displaying an entry field with two columns for “Name” and “Oligonucleotide sequence.”

FIGURE 7.1 A short nucleotide sequence (oligo) can be searched in the input sequence for determining specific RE sites present in the oligos.

Users can also use the following options to make the search for RE sites more stringent by clicking “More Options” (Figure 7.2).

NEBcutter - More options pop-up window displaying multiple options, with a background window highlighting the GenBank account number.

FIGURE 7.2 More options enable the user to make stringent selection of RE sites.

7.3.5 Following options are available under “more options”

7.3.5.1 “Type I and III enzymes” check box

NEBCutter, by default, screens for type II REs (the cleaving location is adjacent to or within the recognition site, independent of methylase, and the REs are magnesium‐dependent). Checking this box will also enable NEBCutter to search for the Type I (cleaving location remote from recognition site; exerts both restriction and methylase activity, and are ATP‐dependent) and Type III (cleaving location similar to Type II; complexed with methylase and are ATP‐dependent) REs.

7.3.5.2 Homing endonucleases

The endonucleases that catalyze the hydrolysis of genomic DNA within the cells synthesizing them. Check this box to include homing endonucleases.

7.3.5.3 Nicking enzymes

These endonucleases cut only one strand of a double‐stranded DNA at a specific recognition site. Checking will include these enzyme sites for screening the input sequence.

7.3.5.4 Check boxes to ignore some of the specific sites in the input sequences

The checked methylase(s) will be ignored while screening for overlapping methylation sensitivity of the enzymes. The methylation‐sensitive restriction enzymes (MSREs) cannot cleave methylated cytosine and, thus, are used to analyze methylated DNA and the methylation status of cytosine residues in CpG sequences:

  1. Ignore CpG methylation
  2. Ignore EcoBI methylation
  3. Ignore Dam methylation
  4. Ignore EcoKI methylation
  5. Ignore Dcm methylation

7.3.5.5 Genetic code to use when searching for ORFs

A drop‐down list of open reading frames (ORFs) is available. The user needs to select the ORF, depending on the input sequence.

7.3.5.6 “Sequence is a fragment” check‐box

Check this when a partial (i.e., an in‐between fragment of a larger string) nucleotide sequence (missing Start or Stop codon) is submitted.

7.3.5.7 “Process this region only” check‐box

Only the specified region is prepared for screening.

7.3.5.8 Set Colors

The user can set colors for different portions of the graphical output, such as Scale, Cut‐Size (blunt, 5′ and 3′ extensions, highlighted ORF and so on).

7.3.5.9 Minimum ORF length to display

Applicable for coding sequence. Minimize the size of the ORF if the coding sequence is shorter.

7.3.5.10 Name of the sequence (optional)

The user needs to provide a name to the sequence being analyzed, as an identifier.

7.3.5.11 Other options

These include a check box for “Disable NEBcutter cookies” and a button to “Delete projects”.

7.3.5.12 “Submit” button

To initiate screening of the input sequence for RE site(s).

7.3.6 Inferring the output

The output page displays the whole input sequence as a single line (if linear sequence option has been selected) with points of RE sites highlighted (Figure 7.3). Each of the RE shown on the RE‐site is hyperlinked (appearing as blue‐colored text), with the page containing detail for the RE. The color schema is displayed at the top‐right part of the output page. Note that the meaning of orange‐colored hash (#) indicates susceptibility of the enzyme to methylation caused by common methylases of E. coli origin. The asterisk (*) symbol indicates susceptibility of the enzyme to CpG methylation.

Image described by caption and surrounding text.

FIGURE 7.3 Result output window of NEBCutter. Details are discussed in the text under the sub‐heading “Inferring the output”.

The page also contains five small panes with hyperlinked words. These have been tabulated and explained in Table 7.1. The explanations have been adopted from the help provided by the NEBCutter tool (http://tools.neb.com/NEBcutter2/help/main_display.html), so sometimes the lines may be verbatim.

TABLE 7.1 Meaning of different terminologies used in NEBCutter (Vince et al., 2003).

Term Meaning
Main Options
New DNA This button, when clicked, opens the initial page
Custom digest To check digestion of input DNA sequence using a set of REs. These enzymes can be further categorized based on the type of the restriction end (blunt or 3’‐overhang or 5’‐overhang) or position of the restriction sites in the target sequence, e.g., REs sites within the input DNA sequence
View sequence To get the input sequence
ORF summary Tabulates following information about the genes that are displayed: coordinates of the genes; length of polypeptide; GenBank protein IDs of the respective gene sequences; single‐cutter REs
Translate GB file The ORF‐finder program of this tool predicts all the non‐overlapping, large open reading frames
Save project Saves the current project in the user’s local disk as a compressed file which can be again uploaded to the site later.
Print To produce a printable file of the current project in PDF, EPS or GIF format.
Availability
All Commercial Displays the REs commercially available from any agencies. The default is NEB‐produced REs.
All Displays all commercial appropriate literature cited, but not commercially available REs.
Display
1, 2 or 3 cutters The default is one cutter REs. The user can also specify displaying two or three cutters, separately.
Alternative/Normal “Alternative” will switch to alternative linear display for two and three cutter REs. Normal will display in the default fashion for all REs together on the scale.
Zoom
Zoom in or Unzoom This enlarges a selected region to a higher resolution (up to base level). More will pop up a window to specify the coordinates to be displayed.
List
0, 1 or 2 cutters The page contains a list of REs as specified by the users (non‐cutter/single‐cutter/double‐cutter). The table contains the name of the enzyme and the RE site (specificity) which can be saved as a text file. The user can also modify the search on some cutters in this page.
All sites Enlists all the RE sites according to their location along the input sequence
Save all sites To save the list of all sites in computer, in *.txt format. The name of the file will be the same as the name of the sequence given by the user.
Flanking enzymes This is very useful for some genetic studies. The user can identify the REs for the regions flanking a target region on the input sequence.

7.4 QUESTIONS

  1. 1. Let us say we are selecting the partial, intergenic spacer sequence of Theileria annulata (NCBI, GenBank Acc. no. AJ538184.1) and Babesia bigemina (AJ538183.1) to detect the presence of the RE site for Cfr131. The objective is to differentiate these two species, based on the presence of the reported RE site (Current Science (2007), 93(12), pp. 1840–1843). Check which of the following sequences harbors the RE site.
  2. 2. Given the vector pBR322 (NCBI GenBank Acc. No. J01749.1), and the insert sequence AJ812216.1, select at least two suitable restriction enzymes which can be used for inserting the insert into the vector sequence at one position.
  3. 3. Diagrammatically depict (using the NEBCutter online tool) the RE sites on the mRNA sequence AY762972.1 for the rare‐cutters SmaI and NotI.
  4. 4. Sickle cell anemia in human occurs due to a missense mutation in the 6th amino acid (Glutamate to Valine: dbSNP Id rs334) in the beta chain of hemoglobin. The mutation is GAG to GTG. Determine which RE can be used to detect the SNP in an individual if the sequence of the amplicon is 5′‐GACACCATGGTGCATCTGACTCCTG[G/T]GGAGAAGTCTGCCGTTACTGCCCTG‐3′.
  5. 5. Beta‐Casein is an important constituent of milk. There are two types of allelic variants, namely, Type A1 and A2 (“CCT” and “CAT”, respectively, at position 350 of both the sequences). Given the sequences of these types of beta casein of bovine milk, determine the specific restriction enzyme that can be used for RFLP study for discerning the two allele‐types:

    > XM_010797953|CSNA2

    A T G C C A T T A A A T A C T A T A T A T A A A C A A C C A C A A A A T C A G A T C A T T A T C C A T T C A G C T C C T C C T T C A C T T C T T G T C C T C T A C T T T G G A A A A A A G G A A T T G A G A G C C A T G A A G G T C C T C A T C C T T G C C T G C C T G G T G G C T C T G G C C C T T G C A A G A G A G C T G G A A G A A C T C A A T G T A C C T G G T G A G A T T G T G G A A A G C C T T T C A A G C A G T G A G G A A T C T A T T A C A C G C A T C A A T A A G A A A A T T G A G A A G T T T C A G A G T G A G G A A C A G C A G C A A A C A G A G G A T G A A C T C C A G G A T A A A A T C C A C C C C T T T G C C C A G A C A C A G T C T C T A G T C T A T C C C T T C C C T G G G C C C A T C C A T A A C A G C C T C C C A C A A A A C A T C C C T C C T C T T A C T C A A A C C C C T G T G G T G G T G C C G C C T T T C C T T C A G C C T G A A G T A A T G G G A G T C T C C A A A G T G A A G G A G G C T A T G G C T C C T A A G C A C A A A G A A A T G C C C T T C C C T A A A T A T C C A G T T G A G C C C T T T A C T G A A A G G C A G A G C C T G A C T C T C A C T G A T G T T G A A A A T C T G C A C C T T C C T C T G C C T C T G C T C C A G T C T T G G A T G C A C C A G C C T C A C C A G C C T C T T C C T C C A A C T G T C A T G T T T C C T C C T C A G T C C G T G C T G T C C C T T T C T C A G T C C A A A G T C C T G C C T G T T C C C C A G A A A G C A G T G C C C T A T C C C C A G A G A G A T A T G C C C A T T C A G G C C T T T C T G C T G T A C C A G G A G C C T G T A C T C G G T C C T G T C C G G G G A C C C T T C C C T A T T A T T G T C T A A G A G G A T T T C A A A G T G A A T G C C C C C T C C T C A C T T T T G A A T T G A C T G C G A C T G G A A A T A T G G C A A C T T T T C A A T C C T T G C A T C A T G T T A C T A A G A T A A T T T T T A A A T G A G T A T A C A T G G A A C A A A A A A T G A A A C T T T A T T C C T T T A T T T A T T T T A T G C T T T T T C A T C T T A A T T T G A A T T T G A G T C A T A A A C T A T A T A T T T C A A A A T T T T A A T T C A A C A T T A G C A T A A A A G T T C A A T T T T A A C T T G G A A A T A T C A T G A A C A T A T C A A A A T A T G T A T A A A A A T A A T T T C T G G A A T T G T G A T T A T T A T T T C T T T A A G A A T C T A T T T C C T A A C C A G T C A T T T C A A T A A A T T A A T C C T T A G G C A T A

    > XM_010806178|CSNA1

    A T G C C A T T A A A T A C T A T A T A T A A A C A A C C A C A A A A T C A G A T C A T T A T C C A T T C A G C T C C T C C T T C A C T T C T T G T C C T C T A C T T T G G A A A A A A G G A A T T G A G A G C C A T G A A G G T C C T C A T C C T T G C C T G C C T G G T G G C T C T G G C C C T T G C A A G A G A G C T G G A A G A A C T C A A T G T A C C T G G T G A G A T T G T G G A A A G C C T T T C A A G C A G T G A G G A A T C T A T T A C A C G C A T C A A T A A G A A A A T T G A G A A G T T T C A G A G T G A G G A A C A G C A G C A A A C A G A G G A T G A A C T C C A G G A T A A A A T C C A C C C C T T T G C C C A G A C A C A G T C T C T A G T C T A T C C C T T C C C T G G G C C C A T C C C T A A C A G C C T C C C A C A A A A C A T C C C T C C T C T T A C T C A A A C C C C T G T G G T G G T G C C G C C T T T C C T T C A G C C T G A A G T A A T G G G A G T C T C C A A A G T G A A G G A G G C T A T G G C T C C T A A G C A C A A A G A A A T G C C C T T C C C T A A A T A T C C A G T T G A G C C C T T T A C T G A A A G C C A G A G C C T G A C T C T C A C T G A T G T T G A A A A T C T G C A C C T T C C T C T G C C T C T G C T C C A G T C T T G G A T G C A C C A G C C T C A C C A G C C T C T T C C T C C A A C T G T C A T G T T T C C T C C T C A G T C C G T G C T G T C C C T T T C T C A G T C C A A A G T C C T G C C T G T T C C C C A G A A A G C A G T G C C C T A T C C C C A G A G A G A T A T G C C C A T T C A G G C C T T T C T G C T G T A C C A G G A G C C T G T A C T C G G T C C T G T C C G G G G A C C C T T C C C T A T T A T T G T C T A A G A G G A T T T C A A A G T G A A T G C C C C C T C C T C A C T T T T G A A T T G A C T G C G A C T G G A A A T A T G G C A A C T T T T C A A T C C T T G C A T C A T G T T A C T A A G A T A A T T T T T A A A T G A G T A T A C A T G G A A C A A A A A A T G A A A C T T T A T T C C T T T A T T T A T T T T A T G C T T T T T C A T C T T A A T T T G A A T T T G A G T C A T A A A C T A T A T A T T T C A A A A T T T T A A T T C A A C A T T A G C A T A A A A G T T C A A T T T T A A C T T G G A A A T A T C A T G A A C A T A T C A A A A T A T G T A T A A A A A T A A T T T C T G G A A T T G T G A T T A T T A T T T C T T T A A G A A T C T A T T T C C T A A C C A G T C A T T T C A A T A A A T T A A T C C T T A G G C A T A

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