(BAHS 201)

(BASIC BIOCHEMISTRY AND

MOLECULAR CELL BIOLOGY)

Session 10– GENE CLONING

Lecturer: Dr. D. K. KONLAN, ESQ

Department of Adult Health, SONM
Contact Information: [email protected]

College of Health Sciences

School of Nursing and Midwifery, DEPARTMENT
OF ADULT HEALTH
2022/2023
 Overview
• DNA can be amplified in vectors. The process of DNA
amplification is known as molecular cloning.
• This session seeks to explain the process of gene cloning
focusing on the various techniques.

Slide 2
 Goals & Objectives
At the end of the session, the student will:
• Explain the process of gene cloning.
• Explain northern and southern blotting techniques
• Describe the way of creating and maintaining DNA
libraries.

Slide 3
 Molecular Cloning.
• To study and manipulate a particular DNA sequence or gene, it is usually necessary
to amplify it.
• The amplification process is called cloning (more precisely-molecular cloning) since
a single recombinant DNA molecule is replicated into million of identical copies.
• This is different from cloning organisms which is the process of taking a single cell
from an organism and using it to regenerate an identical copy of the organism.
• Molecular cloning involves joining a specific DNA fragment to a DNA molecule that
can replicate in a host cell-usually the bacterium E. coli.

• The replicating DNA molecule is called a vector.

• Since the vector can replicate in the bacterial cells, any heterologous (foreign) DNA
covalently joined to the vector will also be replicated.

• Common vectors are Plasmids and bacterial viruses. In eukaryotic cells, vectors can
also be either plasmids or viruses (retroviruses).
 The important features to consider when
choosing a vector are:
• should be small and easy to handle.
• able to generate large amounts of DNA through
replication in an appropriate organism such as
bacteria.
• if necessary can direct expression of a recombinant
protein in appropriate host cell.
• can accommodate DNA of the required size.
• has an appropriate selection marker (antibiotic
resistance)
General procedure for cloning a DNA fragment in a plasmid vector.

B
A

Isolation of DNA fragments from a mixture

The plasmid DNA can then be isolated
from cells in the colony and characterized or
by cloning in a plasmid vector.
otherwise manipulated.
 Some uses of cloning:
• 1. Isolate and characterize a specific gene or mRNA
from a complex mixture (the genome or the total
RNA in a cell).
• 2. Generate probes to detect homologous
sequences-useful in disease diagnosis.
• 3. Express large amounts of protein-useful in
producing therapeutic proteins.
• 4. Generate mutations to develop animal models of
particular diseases.
 DNA Libraries
• Libraries are a complex collection of DNA fragments (for example, the DNA
fragments produced by cutting human DNA with a restriction fragment)
individually inserted into a vector.
• Genomic libraries consist of DNA derived from the genome of an
organism. Genomic libraries therefore contain all elements of the genome-
coding regions, intron, regulatory control regions, regions between gene,
etc.
• cDNA libraries contain double-stranded copies of DNA copied from mRNA.
Thus, a cDNA library only contains the sequences that are present in the
mRNAs. It also contains only those sequences that are expressed-
therefore two cDNA libraries made from two cell types in an organism will
have overlapping but distinct sets of sequences. cDNA is synthesized using
reverse-transcriptase-an enzyme that synthesizes a single strand DNA copy
of an mRNA. The single stranded DNA can then be converted into a
double-stranded molecule using a DNA polymerase and then cloned into a
vector.
Construction of a human Genomic
DNA library.
Genomic library contains all of the genetic information
found in the genome of an organism.
• In the case of human DNA introns, exons and noncoding
regions would all be included.
• Reasons to construct and screen a genomic library include:
• 1. to clone the whole gene including introns and 5’ regulatory
sequences (promoter), for example to study gene expression.
• 2. the gene will be cloned in the context of surrounding DNA
making it possible to move along a chromosome and clone a
new gene whose approximate chromosomal location is
known.
• 3. to make knock-out animal model by replacing the
functional gene with a nonfunctional copy.
The Synthesis of cDNA
 When thinking about constructing or using a
cDNA library remember:
• There are no upstream regulatory sequences
controlling gene expression because they are not
transcribed into the mRNA.
• The cellular source of mRNA is critical! If the cell
does not express the gene , no mRNA, no cDNA and
it won’t be in the library.
• Since there are no introns, cDNA clones can be
“translated” to find open reading frames and predict
protein sequence.
• It is not possible to determine chromosomal
“context” or identify relationship to genes that map
nearby.
The differences between cDNA clones and genomic DNA clones derived from the
same region of DNA
 Nucleic Acid Hybridization
• A common method to detect a specific nucleotide
sequence in a complex mixture such as a library is
hybridization.
• Hybridization is based on the ability of a single-
stranded nucleic acid (probe) to specifically anneal by
base pairing to a complementary sequence present
among many copies of non-complementary
sequences.
• To detect the hybridization product, the probe is
modified so that it can be detected-for example with
radioactive or fluorescent nucleotides.
Membrane hybridization assay
for detecting nucleic acids.

This assay can be used to detect

both DNA and RNA, and the
radiolabeled complementary probe
can be either DNA or RNA.
Separation of DNA fragments of different lengths
by gel electrophoresis

Pulsed-field gel electrophoresis is used to resolve

DNA fragments 20 kb to 10 Mb in length

Visualization of restriction fragments separated by

gel electrophoresis using ethidium bromide and 32P
labeled fragments separated by PAGE respectively.
 Southern and Northern Blotting
• These are procedures in which mixtures of nucleic acids are separated by size and
then probed by hybridization.
• To perform a southern blot, the genomic DNA is first digested to completion with
one or more restriction endonucleases.
• The size separation is carried out by gel electrophoresis in which DNA or RNA is
applied to a porous gel and then subjected to an electric field. The negatively
charged DNA or RNA migrates towards the positive electrode. The smallest
fragments migrate the fastest and therefore at the bottom of the gel.
• The molecules are then transferred to a solid support such as nitrocellulose filter.
• Before the transfer, the DNA gel is treated with an alkaline solution to denature the
DNA, this separates the two complementary strands making them available for
hybridization with the probe.
• The blot is then hybridized to a labeled DNA probe (can be either radioactive or
non radioactive system) to detect the gene or RNA of interest.
Southern blot technique for detecting the presence of specific DNA sequences

In Northern blotting, the total cellular RNA is denatured by treatment with an agent
(e.g formaldehyde) that prevents H-bonding between base pairs, ensuring that all the
RNA molecules have unfolded, linear conformation.
 Southern Blot can be used to Detect:
• large insertion or deletion (50 to 100bp) of chromosomal DNA into the
gene; the restriction fragments which hybridize to the probe will be either
larger or smaller than expected.
• gross gene amplification, this means that rather than the normal number
of 2 copies per diploid cell there may be 5 to 20 or more copies per cell.
(such as the HER-2/neu gene which plays a causative role in the
development of a highly aggressive breast cancer).
• genomic rearrangements caused by chromosomal translocation. This
occurs when two chromosomes actually break and re-join with the wrong
chromosome (an example is the BCR-Abl translocation that plays a
causative role in the development of chronic myeloid leukemia (CML)).
• mutation of a single nucleotide can only be seen on a Southern blot if it
creates or destroys a restriction enzyme site (Hemophilia A is an example).
 A Southern Blot does not detect:
• whether or not a gene is expressed
• point mutations (other than restriction fragment
length polymorphism)
• small deletions and insertions.
 A Northern Blot can detect:
• expression of a specific gene which likely changes
from tissue to tissue (except for “housekeeping”
genes).
• the size of the RNA transcript. An aberrant sized
mRNA could arise as a result of deletion or insertion
in the gene or a chromosomal translocation.
• the relative levels of expression in different samples.
Increased or decreased levels of mRNA are
associated with many disease states
• northern can detect deletions or insertions in genes
as well as splicing defects.
 References
• Berg, J.M., Tymoczko, J.L. & Stryer, L. (2005). Biochemistry 5th
Edition. United States of America. W.H Freeman and
Company
• Devlin T. M. (2010). Textbook of biochemistry with clinical
correlations. 7th edition. Oxford, UK: Wiley- Blackwell.
• Garrett R. H., Grisham C. M. (2012). Biochemistry.5th
edition. New York: Cengage learning Centre.
• Lodish H, Berk A, Kaiser C.A. & Krieger M. (2012). Molecular
cell biology. 7th edition. New York: W. H.
Freeman and Company.
• Nelson, D.L., & Cox, M.M. (2004). Lehnger principles of
biochemistry. 4thedn. New York, NY: W.H. Freeman.
Slide 23