Molecular Biology Experiment: Determining Lambda DNA Restriction Map

This document outlines a molecular biology experiment focused on determining the restriction map of lambda DNA through digestion with restriction enzymes HindIII and XhoI. The experiment aims to understand how these enzymes, both individually and combined, cleave the DNA at specific sites, resulting in different fragment lengths. The results, analyzed using agarose gel electrophoresis, demonstrate the distinct cleavage patterns of each enzyme, allowing for the construction of a restriction map showing the relative positions of the HindIII and XhoI restriction sites on the lambda DNA. The fragment sizes obtained are compared to an idealized map to validate the accuracy of the experimental data. Desklib provides a platform to explore similar solved assignments and past papers for students.

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MOLECULAR BIOLOGY
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Aim:
Determination of the restriction map of lambda DNA produced by the digestion of the
DNA using the restriction enzymes Hind III and XhoI.
Introduction
The restriction enzyme or also called the restriction endonucleases are the bacterial
proteins which play an important role in the defence mechanism of the organisms. The major
activity of the restriction endonucleases are the cleavage of the double stranded DNA
internally which eventually acts as a pair of scissors which cuts both the stands of the DNA
molecule1. This mainly takes place when a foreign DNA like a viral DNA enters the bacteria,
during which the restriction enzyme does its job by cutting the foreign DNA in order to act
like a normal immune system. However the bacteria protects its own DNA by chemically
modifying the DNA which is carried out by the DNA methylase enzyme.
The natural function of these restriction enzymes are taken advantage of in molecular
biology where these enzymes are used to perform various procedures. The two most common
uses are firstly to synthesise DNA molecules which are used in order to create the
recombinant DNA and secondly in order to obtain an estimate of the similarity that is present
in the sequence of nucleotide. Different species of bacteria produce different restriction
enzymes. These are then named according to the strain the have been isolated from. Like for
example, the enzyme EcoRI, which is isolated from Escherichia coli strain R2. In the
following laboratory study, the restriction enzyme will be used in order to determine the
1 Loenen, Wil AM, David TF Dryden, Elisabeth A. Raleigh, Geoffrey G. Wilson, and Noreen E. Murray.
"Highlights of the DNA cutters: a short history of the restriction enzymes." Nucleic acids research 42, no. 1
(2013): 3-19.
2 Gu, Bin, Ying-Chun Zhao, Zhi-Wen Yang, Hong-Tao Li, and Fang-Ping Yu. "HindIII polymorphism in the
lipoprotein lipase gene and hypertensive intracerebral hemorrhage in the Chinese Han population." Journal of
Stroke and Cerebrovascular Diseases 23, no. 6 (2014): 1275-1281.

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restriction map of lambda DNA. This is done by digestion of the lambda DNA using the
restriction enzyme Hind III and XhoI. These two restriction enzymes are used individually as
well as used together. In the given study, the enzyme HindIII cuts the Lambda DNA3. This
helps to deduce the position where the XhoI enzyme cuts the lambda DNA. Additionally this
also allows to identify the restriction map of the DNA along with the position of the
restriction sites which are marked on it.
Discussion
After the laboratory experiment was completed, it was deduced from the study that
the aim of the experiment was met since the restriction fragments could be calculated from
the obtained fragments of nucleotide. The restriction fragment used for the study could cut
the DNA fragments into different lengths, thus producing different lengths of nucleotides.
The results of the experiment showed that the restriction enzyme Hind III cuts the DNA into
several pieces along with the Xho 14. When the two enzymes digests the DNA molecule
separately there are several fragments produced of band fragment size (bp) 23130, 2027,
2322, 9416, 564, 6682 and 4361. Similarly for Xho l, the single digests show 14,125 bp and
12,589 bp. However when these are used together the double digest gives 11,749bp, 9,772bp,
7,762bp, 5,623bp, 3,090bp, 2,570bp and 417bp. Thereby the final size of the double base pair
and digest of Hind III and Xho 1 is of 40, 983bp.
According to the agarose electrophoresis gel results, the lane containing the lamda
DNA with Hind III restriction enzyme, shows 6 fragments of DNA and the lane containing
lambda DNA with Xho 1 restriction enzyme shows two fragments. The final lane containing
3 Pal, Gargi, and Sheela Srivastava. "Cloning and heterologous expression of plnE,-F,-J and-K genes derived
from soil metagenome and purification of active plantaricin peptides." Applied microbiology and
biotechnology 98, no. 3 (2014): 1441-1447.
4 Vranken, Charlotte, Jochem Deen, Lieve Dirix, Tim Stakenborg, Wim Dehaen, Volker Leen, Johan Hofkens,
and Robert K. Neely. "Super-resolution optical DNA Mapping via DNA methyltransferase-directed click
chemistry." Nucleic acids research 42, no. 7 (2014): e50-e50.

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lambda DNA and both restriction enzyme Hind III and Xho 1 gives 7 fragments. This shows
that both the enzymes shows their action both individually and together. The Hind III cleaves
only the palindrome sequences present that is AAGCTT in the presence of the cofactor
Mg2+ via hydrolysis and the Xho I recognise the double-stranded DNA sequence CTCGAG
and cleaves after the C-1 position. However both of these are type II restriction
endonucleases. After the DNA fragments have been generated, each fragment that is
generated by the restriction enzyme which should be sum of the molecular weight of the
lambda DNA. The cleavage sites of the HindIII are smaller in comparison to the Xho 1. The
experimental data that could be compared to the idealized map in order to determine the
accuracy of the available data. Thus from the sizes of the fragments that are obtained, it is
possible to map the relative idealized location of the Hind III and Xho I restriction site.5
5 Sinha, Abhinav, Katie R. Hughes, Katarzyna K. Modrzynska, Thomas D. Otto, Claudia Pfander, Nicholas J.
Dickens, Agnieszka A. Religa et al. "A cascade of DNA-binding proteins for sexual commitment and
development in Plasmodium." Nature 507, no. 7491 (2014): 253.

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References
Loenen, Wil AM, David TF Dryden, Elisabeth A. Raleigh, Geoffrey G. Wilson, and Noreen
E. Murray. "Highlights of the DNA cutters: a short history of the restriction
enzymes." Nucleic acids research 42, no. 1 (2013): 3-19.
Gu, Bin, Ying-Chun Zhao, Zhi-Wen Yang, Hong-Tao Li, and Fang-Ping Yu. "HindIII
polymorphism in the lipoprotein lipase gene and hypertensive intracerebral hemorrhage in the
Chinese Han population." Journal of Stroke and Cerebrovascular Diseases 23, no. 6 (2014):
1275-1281.
Pal, Gargi, and Sheela Srivastava. "Cloning and heterologous expression of plnE,-F,-J and-K
genes derived from soil metagenome and purification of active plantaricin peptides." Applied
microbiology and biotechnology 98, no. 3 (2014): 1441-1447.
Sinha, Abhinav, Katie R. Hughes, Katarzyna K. Modrzynska, Thomas D. Otto, Claudia
Pfander, Nicholas J. Dickens, Agnieszka A. Religa et al. "A cascade of DNA-binding
proteins for sexual commitment and development in Plasmodium." Nature 507, no. 7491
(2014): 253.
Vranken, Charlotte, Jochem Deen, Lieve Dirix, Tim Stakenborg, Wim Dehaen, Volker Leen,
Johan Hofkens, and Robert K. Neely. "Super-resolution optical DNA Mapping via DNA
methyltransferase-directed click chemistry." Nucleic acids research 42, no. 7 (2014): e50-
e50.

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