The document details the process of restriction mapping of bacterial DNA, emphasizing its utility in characterizing DNA molecules and aiding in the engineering of plasmids. It outlines the methods involving restriction enzymes, gel electrophoresis, and interpretation of fragment sizes to construct a restriction map, which is crucial for determining the orientation of DNA inserts in cloning vectors. The document also discusses various applications and the importance of recognition sequences for different restriction enzymes in manipulating DNA.

1. Department of Biochemistry and Molecular Biology
University of Dhaka
MS Practical
Restriction Mapping of Bacterial DNA
Submitted by
Md. Atai rabby
MS

2. Introduction:
A restriction map is a map of known restriction sites within a sequence of DNA. Restriction
mapping requires the use of restriction enzymes. In molecular biology, restriction maps are used as
a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer
genomic DNA. There are other ways of mapping features on DNA for longer length DNA
molecules, such as mapping by transduction (Bitner, Kuempel 1981).
Restriction mapping is a useful way to characterise a particular DNA molecule. It enables us to
locate and isolate DNA fragments for further study and manipulation. The relative location of
different restriction enzyme sites to each other are determined by enzymatic digest of the DNA with
different restriction enzymes, alone and in various combinations.The digested DNA is separated by
gel electrophoresis and the fragment sizes that have been generated are used to build the 'map' of
sites of the fragment. The map lets us know 'where we are' in the linear DNA macromolecule.
One approach in constructing a restriction map of a DNA molecule is to sequence the whole
molecule and to run the sequence through a computer program that will find the recognition sites
that are present for every restriction enzyme known.
Before sequencing was automated, it would have been prohibitively expensive to sequence an entire
DNA strand. Even today sequencing is overkill for many applications. To find the relative positions
of restriction sites on a plasmid a technique involving single and double restriction digests is used.
Based on the sizes of the resultant DNA fragments the positions of the sites can be inferred.
Restriction mapping is very useful technique when used for determining the orientation of an insert
in a cloning vector, by mapping the position of an off-center restriction site in the insert (Dale, Von
Schantz, 2003).
Method : The experimental procedure first requires an aliquot of purified DNA for each digest to
be run. Digestion is then performed with each enzyme(s) chosen. The resulting samples are
subsequently run on an electrophoresis gel, typically on agarose gel.
The first step following the completion of electrophoresis, is to add up the sizes of the fragments in
each lane. The sum of the individual fragments should equal the size of the original fragment, and
each digest's fragments should also sum up to be the same size as each other. If fragment sizes do
not properly add up, there are two likely problems. In one case, some of the smaller fragments may
have run off the end of the gel. This frequently occurs if the gel is run too long. A second possible
source of error is that the gel was not dense enough and therefore was unable to resolve fragments
close in size. This leads to a lack of separation of fragments which were close in size. If all of the
digests produce fragments that add up one may infer the position of the REN (restriction
endonuclease) sites by placing them in spots on the original DNA fragment that would satisfy the
fragment sizes produced by all three digests.

3. Example : For example the most common application of restriction mapping is presented:
Determining the orientation of a cloned insert. This method requires that restriction maps of the
cloning vector and the insert are already available.
If you know of a restriction site placed towards one end of the insert you can determine the
orientation by observing the size of the fragments in the gel. Oftentimes the orientation of inserts is
important and this technique is used to screen for the correct orientation.
In this example the orientation of an insert cloned with EcoRI will be found.
Digests
1: EcoRI
2: HindIII
3: EcoRI + HindIII
Resultant Fragments: approximate sizes
 1: 3 kb, 5 kb
 2: 2 kb, 6 kb
 3: 2 kb, 1 kb, 5 kb,
Hypothetical Multiple Cloning Site of Vector
5'-----HindIII-EcoRI----3'
Discussion : The EcoRI digest excises the insert yielding fragments of 3 kb and 5 kb. These are the
sizes of the insert and vector backbone respectively. This is expected since the size of both the
insert and vector are known beforehand. The presence of the insert is confirmed.
There is a known HindIII site off-center in the 3 kb insert. It is 2 kb away from one end (end A),
and 1 kb away from the other end (end B). The HindIII digest of your clone yields fragments of 2
kb and 6 kb. The 2 kb fragment is exclusively the insert sequence and the 6 kb fragment is 1 kb of
insert sequence attached to 5 kb of vector sequence. This means that the insert was cloned in an A
to B orientation as opposed to B to A which would yield fragments of 7 kb and 1kb.

4. resultant map
Fig : - Resultant map
REAGENTS
1. Buffer 2:
 10X final concentration
 50mM Tris pH
 10mM MgCl2
 100mM NaCl
2. PCR water
3. DNA sample
4. Restriction Enzymes:
 EcoR I
 Hind III
 Bam HI

5. Table : Amount of Restriction Enzymes in the samples.
Tube
No
Buffer
µL
PCR H2O
µL
Eco RI
µL
Hind III
µL
Bam HI
µL
Eco RI +
Hind III
µL
Eco RI + Hind
III + Bam HI
µL
1 1 6.0
2 1 5.5 0.5
3 1 5.5 0.5
4 1 5.5 0.5
5 1 5 0.5+0.5
6 1 4.5 0.5+0.5+0.5
Disscussion
The purpose of this lab is to better understand some of the properties of plasmids and the use of restriction
enzymes in biotechnology. The uses of the reagents are outlined below:
 PCR water: To make the volume of the tubes equal.
 Loding dye: To increase the density of the sample and enable us to see the
progress of the sample through the gel.
 Agarose gel: Generally 0.8% agarose gel is used. As the expire date was over, 1.2%
agarose gel was used to compansate the gel breaking problem.
Restriction mapping is the process of obtaining structural information on a piece of DNA by the use
of restriction enzymes.

6. Restriction enzymes: Restriction enzymes are enzymes that cut DNA at specific recognition
sequences called "sites." They probably evolved as a bacterial defense against DNA bacteriophage.
DNA invading a bacterial cell defended by these enzymes will be digested into small, non-
functional pieces. The name "restriction enzyme" comes from the enzyme's function of restricting
access to the cell. A bacterium protects its own DNA from these restriction enzymes by having
another enzyme present that modifies these sites by adding a methyl group. For example, E.coli
makes the restriction enzyme Eco RI and the methylating enzyme Eco RI methylase. The methylase
modifies Eco RI sites in the bacteria's own genome to prevent it from being digested.
Restriction enzymes are endonucleases that recognize specific 4 to 8 base regions of DNA. For
example, one restriction enzyme, Eco RI, recognizes the following six base sequence:
5' . . . G-A-A-T-T-C . . . 3'
3' . . . C-T-T-A-A-G . . . 5'
A piece of DNA incubated with Eco RI in the proper buffer conditions will be cut wherever this
sequence appears. As you can see, this site is palindromic; that is, reading the upper strand from 5'
to 3' is the same as reading the lower strand from 5' to 3'. As a result, each strand of the DNA can
self-anneal and the DNA forms a small cruciform structure:
Figure 1
All restriction enzyme sites are palindromic. This structure may help the
enzyme to recognize the sequence that it is designed to cut.

7. There are hundreds of restriction enzymes that have been isolated and each one recognizes its own
specific nucleotide sequence. Sites for each restriction enzyme are distributed randomly throughout
a particular DNA stretch. Digestion of DNA by restriction enzymes is very reproducible; every time
a specific piece of DNA is cut by a specific enzyme, the same pattern of digestion will occur.
Restriction enzymes are commercially available and their use has made manipulating DNA very
easy.
Restriction Mapping: Restriction mapping involves digesting DNA with a series of restriction
enzymes and then separating the resultant DNA fragments by agarose gel electrophoresis. The
distance between restriction enzyme sites can be determined by the patterns of fragments that are
produced by the restriction enzyme digestion. In this way, information about the structure of an
unknown piece of DNA can be obtained. An example of how this works is shown below. You have
isolated a clone in pBluescript (look at bacterial transformation lab again to see its restriction map).
You know how big the pBluescript portion of the plasmid is (3.0 kilobases) and what restriction
enzymes are present in the plasmid (because you have its restriction map from the company that
sold you the plasmid). You also know that the insert is 2.0 kb long and that it is inserted the Eco RI
site. Your task is to find out more information about the insert:
Figure 2
At this point, we would digest plasmid with an enzyme that you know is in the pBluescript plasmid.
For example, you know that there is only one Bam HI site in pBluescript, and it is in the multiple
cloning site next to the Eco RI site (figure 2). If we digest this plasmid with Bam HI, there are two
possibilities: 1) There are no Bam HI sites in the insert. If this is the case, when we run this
digestion on a gel we will see only one DNA fragment, and it will be 5.0 kb long (3.0 kb of

8. pBluescript DNA and 2.0 kb of insert DNA). 2) There is a Bam HI site in the insert. If this is the
case, then the enzyme will cut the circular plasmid in two places, in the pBluescript part of the
plasmid and in the insert. In this case, we will end up with two fragments of DNA. One will be
pBluescript with some of the insert still attached and the other will be just insert. The sizes of the
two fragments (determined by
electrophoresis) will tell us
where the site is. These two
possibilities are shown in
<< figure 3
In the second case, where there is a site in the
insert, the gel might look like this:
Figure4
In this case, we learn two pieces of information:
1) that there is a Bam HI site in the insert, and 2)
where the site is in relation to the one end of the
insert. When the Bam HI digestion is separated
on an agarose gel, the sizes of the two fragments
can be determined. In the above gel, the
fragments are 3.6 kb and 1.4 kb.

9. Therefore, we know that the Bam HI site is 1.4 kb away from the right hand side of the insert
(figure 5). In this way, you have "mapped" the Bam HI site:
Figure 5
By testing the insert for the presence and location of
sites of many different restriction enzymes, a
"restriction map" of the clone is made. This will give us
important structural information on the insert.
Recognition site : Restriction enzymes recognize a specific sequence of nucleotides and produce a
double-stranded cut in the DNA. While recognition sequences vary between 4 and 8 nucleotides,
many of them are palindromic, which correspond to nitrogenous base sequences that read the same
backwards and forwards. In theory, there are two types of palindromic sequences that can be
possible in DNA. The mirror-like palindrome is similar to those found in ordinary text, in which a
sequence reads the same forward and backwards on the same DNA strand (i.e., single stranded) as
in GTAATG.
5'-GTATAC-3'
::::::
3'-CATATG-5'
A palindromic recognition site reads the same on the reverse strand as it does on the forward
strand
The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but
the forward and backward sequences are found in complementary DNA strands (i.e., double
stranded) as in GTATAC (Notice that GTATAC is complementary to CATATG).

10. The inverted repeat is more common and has greater biological importance than the mirror-like.
EcoRI digestion produces "sticky" ends,
whereas SmaI restriction enzyme cleavage produces "blunt" ends
Recognition sequences in DNA differ for each restriction enzyme, producing differences in the
length, sequence and strand orientation (5' end or the 3' end) of a sticky-end "overhang" of an
enzyme restriction.
Different restriction enzymes that recognize the same sequence are known as neoschizomers. These
often cleave in different locales of the sequence. Different enzymes that recognize and cleave in the
same location are known as isoschizomers.
Uses of Restriction Mapping : Restriction map information is important for many techniques used
to manipulate DNA. One application is to cut a large piece of DNA into smaller fragments to allow
it to be sequenced. Genes and cDNAs can be thousands of kilobases long (megabases - Mb);
however, they can only be sequenced 400 bases at a time. DNA must be chopped up into smaller
pieces and subcloned to perform the sequencing. Also, restriction mapping is an easy way to
compare DNA fragments without having any information of their nucleotide sequence. For
example, you may isolate two clones for a gene that are 8 kb and 10 kb long. You know that they
overlap, because the procedure you used to isolate them told you that they have sequences in
common. A restriction map can tell you how much they overlap by:

11. Figure 6
From the restriction map information, we can tell which parts of the two clones are identical and
which parts are different. The parts of the clones that overlap are identical.