2017

Biyoinformatics
Homework - 5
Using REBASE and NEBcutter Web
Applications to Find and Digestion
DNA and Plasmid Restriction
Enzymes
Gebze Technical Universirty

EBRU AKHARMAN
142204026
24.12.2017
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Using REBASE and NEBcutter Web Applications to Find and
Digestion DNA and Plasmid Restriction Enzymes
HW5
Ebru AKHARMAN - 142204026, Gebze Technical University, Turkey

AIM:
REBASE is a comprehensive and fully curated database of information about the components of restriction-
modification (RM) systems. It contains fully referenced information about recognition and cleavage sites for both
restriction enzymes and methyltransferases as well as commercial availability, methylation sensitivity, crystal
and sequence data. All genomes that are completely sequenced are analyzed for RM system components, and with
the advent of PacBio sequencing, the recognition sequences of DNA methyltransferases (MTases) are appearing
rapidly. Thus, Type I and Type III systems can now be characterized in terms of recognition specificity merely by
DNA sequencing.

NEBcutter is a program available via a web server ( http://tools.neb.com/NEBcutter ) that will accept an input
DNA sequence and produce a comprehensive report of the restriction enzymes that will cleave the sequence. It
produces a variety of outputs including restriction enzyme maps, theoretical digests and links into the restriction
enzyme database, REBASE (http://www.neb.com/rebase). Importantly, its table of recognition sites is updated
daily from REBASE and it marks all sites that are potentially affected by DNA methylation (Dam, Dcm, etc.). Many
options exist to choose the enzymes used for digestion, including all known specificities, subsets of those that are
commercially available or sets of enzymes that produce compatible termini.
INTRODUCTION:
REBASE: including their recognition specificities and their
sensitivity to DNA methylation . In the 1970s they
Many of you frequently use REBASE to obtain up-to- were used extensively to provide physical maps of
date information about restriction enzymes, the
small DNAs and, in the 1980s, were used to map large
workhorses of biotechnology research. The
Restriction Enzyme dataBASE (REBASE) was created DNAs. As methods for the determination of DNA
in the 1970s by New England Biolabs and is patented sequence improved, it became convenient to search
by Nobel Laureate Sir Richard Roberts. REBASE those sequences for potential restriction enzyme
provides a wealth of information about restriction cleavage sites since that would permit the facile
enzyme type, recognition sequence, and cut site. It further manipulation of specific fragments. In fact,
also contains information about methyltransferases, until the advent of the polymerase chain reaction
crystal structures, and much more.
(PCR), they provided the most convenient way to
However, the problem with REBASE is its challenging manipulate individual genes and move them from
user interface. When you visit the site, you see an one vector to another. For a while, it seemed that the
array of image buttons on the home screen. Once you
ability of PCR to permit precise amplification of
become familiar with the options on this page and
understand how to navigate REBASE, you will be able individual stretches of DNA might render the use of
to complete projects much faster with increased restriction enzymes obsolete. However, they merely
efficiency and accuracy. found new utility by then serving as diagnostic
reagents to show that DNA constructs had been made
NEBcutter:
correctly. They still provide one of the cheapest and
The Type II restriction enzymes are among the most most convenient ways to characterize DNA
valuable tools available to researchers in molecular constructs. They have also found use in analyzing the
biology. These enzymes recognize short DNA genomes of higher organisms using restriction
sequences (4–8 nucleotides) and cleave at, or close fragment length polymorphisms (RFLPs) as physical
to, their recognition sites. A comprehensive database markers or by directly detecting the presence of
(REBASE) contains information about these enzymes single nucleotide polymorphisms (SNPs).

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Restriction Enzyme:

Preparation of DNA for traditional cloning methods is dependent upon restriction enzyme digestion to generate
compatible ends capable of being ligated together. The DNA to be cloned can vary widely, from genomic DNA
extracted from a pure bacterial culture or a mixed population, to a previously cloned gene that needs to be moved
from one vector to another (subcloning). Restriction enzymes can also be used to generate compatible ends on
PCR products. In all cases, one or more restriction enzymes are used to digest the DNA resulting in either non-
directional or directional insertion into the compatible plasmid.

Genomic DNA, regardless of the source, is typically digested with restriction enzymes that recognize 6-8
consecutive bases, as these recognition sites occur less frequently in the genome than 4-base sites, and result in
larger DNA fragments. The desired insert size for the clone library determines which enzymes are selected, as well
as the digestion conditions. Most often, a serial dilution of the selected restriction enzyme(s) is used to digest the
starting material and the desired insert size range is isolated by electrophoresis followed by gel extraction of the
DNA. This method of preparation provides DNA fragments of the desired size with ends compatible to the selected
vector DNA.

Subcloning requires the use of 1-2 restriction enzymes that cut immediately outside the insert fragment without
cutting within the insert itself. Restriction enzymes that have a recognition site within the multiple cloning site
(MCS) are commonly used since they do not cut elsewhere in the vector DNA and typically produce two easily
resolved DNA fragments. The gene of interest is most commonly subcloned into an expression vector for improved
protein expression and/or addition of a purification tag. In this case, it is essential that the gene be inserted in the
correct orientation and in frame with the transcription promoter.

The Polymerase Chain Reaction (PCR) is commonly used to amplify a gene or DNA fragment of interest, from any
source of DNA, to be cloned. In order to generate compatible ends, it is common to add restriction sites to the 5’
end of both PCR primers. When adding restriction sites to a PCR primer, it is recommended to include 6 bases
between the recognition site and the 5’ end of the primer. These additional bases provide sufficient DNA for the
restriction enzyme to bind the recognition site and cut efficiently. When selecting a restriction site(s) to add to the
primers, it is important to determine which site(s) will be compatible with your selected vector, whether
directional cloning is desired and, most importantly, confirm that the recognition site(s) does not occur within the
gene or DNA fragment.

Image 1: Nucleotide Sequence of Bacillus alcalophilus 16S rRNA gene sequence

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Image 2: NEBcutter Page in ReBase Page

The DNA sequence is pasted to the indicated site. Then select "All commercially available specificities". Each
sequence used to avoid confusion among the sequences being studied is named. You can see these sequences later.
Then click "Submit" button.

Image 3: Linear Sequence of Bacillus alcalophilus

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This picture shows a map of the enzymes that once cut DNA of Bacillus alcalophilus. DNA indicates the ratio of AT
and CG contained. With the "Custom Digest" option, the cut points of the desired enzyme are visible. In addition,
enzymes are classified according to cutting numbers.

For Enzyme of Sau3AI:

Image 4: Sau3AI Enzyme

The "Custom Digest" option is selected and the enzyme is selected from the page. Then click the "Digest" button.

Image 5: Custom Digest For Bacillus alcalophilus

It is observed that the enzyme Sau3AI has 5 cutting points on Bacillus alcalophilus. From this page, gel image of the
enzyme is reached.

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Image 6: Gel Image of Enzyme

The marker for the enzyme gel image should contain the moving DNA fragments. The piece of DNA must walk
ahead of the marker. The marker and the DNA fragment should be close to one another. This image shows the
length of the restricted DNA and identifies in which areas it is cut off.

Image 7: Cut Positions

From this page you can see the cutting positions of the enzyme.

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For Enzyme of MseI:

Image 8: MseI Enzyme

The "Custom Digest" option is selected and the enzyme is selected from the page. Then click the "Digest" button.

Image 9: Custom Digest For Bacillus alcalophilus

It is observed that the enzyme MseI has 5 cutting points on Bacillus alcalophilus. From this page, gel image of the
enzyme is reached.

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Image 10: Gel Image of Enzyme

The marker for the enzyme gel image should contain the moving DNA fragments. The piece of DNA must walk
ahead of the marker. The marker and the DNA fragment should be close to one another. This image shows the
length of the restricted DNA and identifies in which areas it is cut off.

Image 11: Cut Positions

From this page you can see the cutting positions of the enzyme.

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For Enzyme of MboII:

Image 12: MboII Enzyme

The "Custom Digest" option is selected and the enzyme is selected from the page. Then click the "Digest" button.

Image 13: Custom Digest For Bacillus alcalophilus

It is observed that the enzyme MboII has 4 cutting points on Bacillus alcalophilus. From this page, gel image of the
enzyme is reached.

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Image 13: Gel Image of Enzyme

The marker for the enzyme gel image should contain the moving DNA fragments. The piece of DNA must walk
ahead of the marker. The marker and the DNA fragment should be close to one another. This image shows the
length of the restricted DNA and identifies in which areas it is cut off.

Image 14: Cut Positions

From this page you can see the cutting positions of the enzyme.

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For Xmn1:

Image 15: MboII Enzyme

The "Custom Digest" option is selected and the enzyme is selected from the page. Then click the "Digest" button.

Image 16: Custom Digest For Bacillus alcalophilus

It is observed that the enzyme Xmn1 has 2 cutting points on Bacillus alcalophilus. From this page, gel image of the
enzyme is reached.

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Image 17: Gel Image of Enzyme

The marker for the enzyme gel image should contain the moving DNA fragments. The piece of DNA must walk
ahead of the marker. The marker and the DNA fragment should be close to one another. This image shows the
length of the restricted DNA and identifies in which areas it is cut off.

Image 18: Cut Positions

From this page you can see the cutting positions of the enzyme.

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For pBR322:

Image 19: Select Plasmid

Select the desired plasmids "plasmid" can be selected from the fields "Allen commercially available specificities"
field should be marked. Each plasmid used to avoid confusion among the plasmids being studied is named. You can
see these plasmids later. Then click "Submit" button.

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Image 20: Plasmid Map With One Cutter Restriction Enzyme

This picture shows a map of the enzymes that once cut plasmid. plasmid indicates the ratio of AT and CG
contained. With the "Custom Digest" option, the cut points of the desired enzyme are visible. In addition, enzymes
are classified according to cutting numbers.

Image 21: Other Options

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Image 22: The Image of One Cutter Enzyme List

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For AvaII Enzyme:

Image 23: AvaII Enzyme

The "Custom Digest" option is selected and the enzyme is selected from the page. Then click the "Digest" button.

Image 24: Plasmid Map With AvaII Restriction Enzyme

It is observed that the enzyme AvaII has 8 cutting points on Bacillus alcalophilus. From this page, gel image of the
enzyme is reached.

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Image 25: Image of Gel

The marker for the enzyme gel image should contain the moving DNA fragments. The piece of DNA must walk
ahead of the marker. The marker and the DNA fragment should be close to one another. This image shows the
length of the restricted DNA and identifies in which areas it is cut off.

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For DNA Cloning:

Image 26: Enzyme of Clonning

The enzyme used for cloning should never cut the DNA sequence.

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Image 27: One Cutter Enzymes of pBR322

Enzymes used for cloning should never cut DNA sequences, but plasmid should be cut at least once. Depending on
the type of work, the plasmid can be cut more than once. The nucleotide sequence of Bacillus alcalophilus has 111
enzymes that do not cur and 48 enzymes that cut pBR322 1 time. There are 27 enzymes for cloning.

Common enzyme is below.

14: NdeI
1: AhdI 15: NheI
2: AlwNI 16: NruI
3: AseI 17: PciI
4: BamHI 18: PflMI
5: BmtI 19: PfoI
6: BsgI 20: PstI
7: BsmBI 21: Pvu II
8: BspDI 22: Sa1I
9: BspQI 23: SapI
10: BstZ17I 24: ScaI
11: ClaI 25: SgrAI
12: EcoRV 26: SspI
13: Hind III 27: Tth111I

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RESOURCES:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC168933/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383893/

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