Molecular Techniques Discussion 2022

Running Head: Molecular Techniques 1
Traditional and Modern Molecular Techniques in Interferon Gene Technology
Name
Course
Institution
Date

Molecular Techniques 2
Table of Contents
Abstract................................................................................................................................................3
Introduction.........................................................................................................................................3
Summary..............................................................................................................................................4
Methods Used.......................................................................................................................................5
mRNA Isolation, Preparation, and Translation............................................................................5
Immunoprecipitation Assay and (SDS-PAGE).............................................................................5
Interferon Assays and Cloning.......................................................................................................6
Advantages and Limitations of the Methods Used........................................................................6
Proposal for Modern Molecular Techniques.....................................................................................7
Isolation of Interferon Gene............................................................................................................7
Cloning and Expression..................................................................................................................8
Conclusion............................................................................................................................................8

Molecular Techniques 3
Abstract
Molecular methods have evolved over time due to advancements in technology. Early
techniques were cumbersome, time-consuming and expensive. The advent of polymerase
chain reaction changed the entire field of molecular biology in that a gene can be isolated and
amplified in one test-tube for a short time. Other advances in the molecular methods include
the advent of more sequencing methods such as the Oxford Nanopore, which are faster and
efficient, compared to earlier techniques such as Sanger sequencing. In this report, we discuss
the methods used for the isolation, and cloning of the human interferon gene [1] and compare
them to modern techniques that could have made the process more efficient.
Introduction
Interferons are a group of proteins usually expressed to bolster the immune system in
fighting viruses [2]. The production of various types of interferons is the body’s most rapid
and important defense system against viral pathogens. The proteins also combat bacterial
infections and may promote or inhibit cell differentiation and division. These benefits of
interferons made early researchers look for means of expressing the proteins in-vitro. The in-
vitro methods try to mimic in-vivo synthesis to produce viable large amounts of proteins that
can be used for commercial purposes. Methods used to produce proteins such as interferons
have evolved.
Protein synthesis starts by transcription of the gene containing the information to
produce a messenger RNA. The mRNA undergoes post-transcriptional modifications before
being transferred to the ribosomes by transfer RNA. In the ribosomes, the codons are read
and translated into amino acids. Each codon codes for a specific amino acid. Many amino
acids joined together to form a polypeptide that undergoes post-translational modification to
produce a fully functional and active protein [3]. The whole of this process can be replicated
in-vitro using certain molecular techniques. In this paper, we will review a study carried out
by Weissenbach and others in 1980 and compare the molecular methods they used to modern

Molecular Techniques 4
techniques. In their research, they expressed two mRNAs that code for human fibroblast
interferons.
Summary
The study carried out by Weissenbach and others [1] involved in-vitro translation of
two mRNAs coding for human fibroblast interferon and cloning in Escherichia coli. They
isolated the two mRNAs which they identified using size fractionation and cloned them in the
pBR322 plasmid vector. The first step aimed to isolate and confirm the mRNA isolates.
Confirmation of the presence of mRNA was done using absorbance assays and translation in
diploid fibroblasts to get two types of interferon proteins; Hu IFN-β1 and Hu IFN-β2. Gel
electrophoresis of the cell lysate was used to determine the presence of the proteins based on
their molecular weight. The following results were obtained. The presence of two
independent mRNAs was further confirmed by treating the SV80 cells using poly(rl.rC) and
cycloheximide which induces the transcription of 11S and 14S mRNAs respectively. 11S
mRNA is known to code for the 20,000 Dalton interferons while the 14S mRNA code for the
23,000 Dalton protein [1].
Cytoplasmic mRNA was successfully isolated from diploid fibroblasts induced after a
4-hour incubation period using 50μg/ml of cycloheximide and 100μg/ml of poly(rl.rC).
absorbance assay and subsequent plotting of the curves revealed two peaks that were
consistent with the presence of 14S and 11S interferon mRNA [1]. Polyacrylamide gel
electrophoresis showed that two protein products of between 23,000-26,000 and 19,000-
20,000 Daltons are obtained from induced mRNA compared to un-induced transcripts. The
two mRNAs corresponded to the 23,000 and 20,000 Dalton polypeptides respectively. When
the cells were treated using cycloheximide a 10-20% increase in 23,000 Dalton protein, the
products of 14S mRNA. Treatment with Poly(rl.rC) coincided with an elevation of the
amount of 20,000 Dalton protein thus 11S mRNA. The team further found out that the 20,000

Molecular Techniques 5
Dalton protein a product of 11S mRNA as the major fibroblast interferon. Therefore, the
group concluded that the 11S mRNA is the Hu IFN-β1 and isolated cDNA clones to
determine the nature of the 14S mRNA which they designated as Hu IFN-β2 [1].
Methods Used
mRNA Isolation, Preparation, and Translation
Various molecular techniques were used in the research study carried out by
(Weissenbach et al., 1980). The team had to first isolate, and identify the mRNAs before
proceeding with the other steps. Messenger RNA was purified from fibroblasts grown.
Isolation involved using Poly(rl.rC) to induce fibroblast cells. Poly(A+) mRNA was isolated
and Purified using sucrose gradient purification method [1]. The mRNA was then translated
in rabbit reticulocyte lysates. The antibodies to the antibodies were produced in rabbits and
later used for immunoprecipitation of the translated proteins. The translated products were
methionine -labeled with A-Sepharose [1].
Immunoprecipitation Assay and (SDS-PAGE)
Immunoprecipitation assay is a technique that was used to precipitate the translated
products from anti-sera products. The method uses antibodies to isolate specific proteins or
antigens from the solution. The isolated antigens are analyzed using Sodium Dodecyl
Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). In this paper, rabbits were
immunized with partially purified interferon extracted from fibroblasts. The antibodies
produced were then used for the immunoprecipitation assay to elute translated interferons.
Immunoprecipitation involves incubating an antibody with antigen to facilitate antigen-
antibody interaction. An immobilized secondary antibody against the primary antibody is
then used to isolate the Ab-Ag complex from the solution [1].
SDS-PAGE is a molecular technique that is used to isolate proteins based on their
molecular weight and size. Sodium dodecyl sulfate is a detergent that denatures proteins that
eliminates the influence of shape and charge and thus the proteins are separated based on

Molecular Techniques 6
their molecular weights. After the translation of the FS11 mRNA in the reticulocyte cell-free
system, the resulting interferon was immunoprecipitated using anti-interferon antibodies
produced in immunized rabbits. The resulting proteins were then run in polyacrylamide gels
to determine the molecular weight of the isolated proteins. After electrophoresis, the gels
were exposed to fluorography and autoradiographed using X-ray films [1].
Interferon Assays and Cloning
The assays were done by checking the activity of the isolated proteins against viral
plaques. (2’,5’) Oligo(A) Synthetase was also used to assay the interferons. Reverse
transcriptase from avian myeloblast virus was used to get cDNA from the mRNA prepared in
the sucrose gradient. Double strand cDNA was then made with DNA polymerase. Restriction
endonuclease was used to linearize pBR322 plasmid which was annealed to the cDNA and
transformed into E-Coli cells grown in L broth agar with tetracycline. Nitrocellulose
technique was used to hybridize the plasmid digested with EcoR1 to poly(A+) mRNA that
had been induced from the fibroblast cells. After boiling to elute the hybridized mRNAs
rabbit tRNA was added and ethanol precipitation carried out [1].
Advantages and Limitations of the Methods Used.
Cloning was done in E-Coli using the pBR322 plasmid vector. These plasmids are
produced in large copy numbers in the bacteria since they are not linked to cell division. This
ensured that large numbers of mRNA were produced. The vector has multiple cloning sites.
Disadvantages of this cloning system include the limited size of a gene that can be inserted
and laborious and time-consuming screening time. The plasmids can move to other cells if a
conjugation factor is present and can easily get lost. The copy number obtained is not as high
as in modern cloning systems [4].
The immunoprecipitation (IP) technique is ideal for the isolation and enrichment of
the proteins on small-scale. The technique is also relatively fast and easy compared to other
molecular techniques such as affinity chromatography [5]. The technique is also compatible

Molecular Techniques 7
to use for other downstream techniques such as mass spectrometry and western blotting. This
is because the native structure of the protein is maintained. IP has limitations in that it is not
effective in analyzing transient and low-affinity proteins. The technique also requires other
validation assays to identify specific proteins because it can’t isolate third proteins
sandwiched between the desired protein and the antibody. IP is limited by the availability of
antibodies that are specific to the desired proteins. The technique is also not suitable for high
throughput screening [6].
SDS-PAGE strengths include the separation of proteins based on their molecular
weight [6]. The procedure also requires small sample amounts while being highly sensitive
since it can isolate compounds with up to 2% difference in mass. The gel is also chemically
stable. The technique's weaknesses include the denaturation of proteins that cannot be used
for downstream processes. The technique has a low throughput in that data is not produced
rapidly because it requires more time to make the gels and run in an electric field.
Acrylamide used in the technique is a powerful neurotoxin poison [7].
Proposal for Modern Molecular Techniques.
Isolation of Interferon Gene.
Earlier DNA extraction was long and cumbersome. In modern times I would use a
DNA extraction micro kit from Qiagen QIAamp [8]. In this process, silica beads in a column
bind DNA in a high chaotropic salt environment. A cell lysate is placed inside a spin column
that is placed in a tube. The unwanted cellular components such as divalent cations, for
example, magnesium, proteins, and lipids are removed using multiple buffer washes. DNA
bound to the membrane of the spin column is then eluted using Tris-EDTA buffer or sterile
water. The isolated DNA is genomic DNA. To get specific interferon genes I would proceed
to use the Polymerase Chain Reaction (PCR) technique using gene-specific primers [9].
PCR is an in-vitro DNA replication technique whereby DNA polymerase is used to
produce numerous copies of a gene [10]. Polymerases require a primer to initiate DNA

Molecular Techniques 8
elongation. By using chemically synthesized primers that are complementary to the human
interferon gene I will demarcate the regions within the isolated DNA that should be
amplified. To amplify the genes, I will add the necessary components required for replication
in the PCR tube. PCR isolation and amplification of the specific interferon genes from the
whole genomic DNA removes the need for using plasmids to amplify the copies produced.
This technique also replaces the immunoprecipitation techniques and the use of SDS-PAGE
to determine whether the isolated RNA is from human interferon genes.
Cloning and Expression
Cloning and expression of the interferon gene will be done in an antibiotic-free
environment using E.coli SE1–pStaby expression vector system [11]. This is because
traditional cloning and expression vectors of interferon proteins in E-coli cells for biomedical
and commercial uses have limitations. For instance, the low yield of target protein due to
[12] plasmid instability in high-density cell cultivation), and antibiotics used are a major
concern. The isolated and amplified genes will be cut using NotI and NdeI restriction
enzymes and inserted into the pET-23a(+) cloning vector. The resulting plasmid will be
transformed into competent E-coli cells through electroporation. Cultivation of the
recombinant cells will be carried out in a laboratory-scale bioreactor before transfer to the
industrial scale.
Conclusion
Traditional or earlier methods used to produce interferon proteins have more
limitations compared to modern molecular techniques. The procedure used to isolate the
interferon mRNA gene was lengthy and cumbersome compared to the procedure that I would
use today. The amount of materials is more in traditional methods which makes them more
expensive compared to modern molecular techniques. Modern molecular techniques such as
PCR have made molecular processes more efficient by reducing the period that was initially
required to isolate and amplify genes.

Molecular Techniques 9
References
Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., ... & Revel,
M. (1980). Two interferon mRNAs in human fibroblasts: in vitro translation and
Escherichia coli cloning studies. Proceedings of the National Academy of Sciences,
77(12), 7152-7156.
Ott, T. L., & Ealy, A. D. (2018). Interferons. In Encyclopedia of Reproduction (pp. 412-416).
Elsevier.
Durairaj, G., Malik, S., & Bhaumik, S. R. (2017). Eukaryotic gene expression by RNA
polymerase II. Gene Regulation, Epigenetics, and Hormone Signaling, 1, 1-28.
Riggs, P. D. (2018). Overview of Protein Expression Vectors for E. coli. Current Protocols
Essential Laboratory Techniques, 17(1), e23.
Lin, J. S., & Lai, E. M. (2017). Protein–Protein Interactions: Co-Immunoprecipitation.
In Bacterial Protein Secretion Systems (pp. 211-219). Humana Press, New York, NY.
Darnell, J. C., Mele, A., Hung, K. Y. S., & Darnell, R. B. (2018). Immunoprecipitation and
SDS-PAGE for cross-linking immunoprecipitation (CLIP). Cold Spring Harbor
Protocols, 2018(12), pdb-prot097956.
Pathak, N. (2017). SDS-PAGE Electrophoresis for Protein Separation–An Overview.
International Journal of Chemical Separation Technology, 3(1), 29-38.
Elkins, K. M. (2013). Chapter 4-DNA extraction. Forensic DNA Biology. Academic Press,
San Diego, 39-52.
Chacon-Cortes, D., & Griffiths, L. R. (2014). Methods for extracting genomic DNA from
whole blood samples: current perspectives. Journal of Biorepository Science for
Applied Medicine, 2014(2), 1-9.

Molecular Techniques 10
Green, M. R., & Sambrook, J. (2019). Polymerase chain reaction. Cold Spring Harbor
Protocols, 2019(6), pdb-top095109.
Pal, D., Tripathy, R. K., Teja, M. S., Kumar, M., Banerjee, U. C., & Pande, A. H. (2018).
Antibiotic-free expression system for the production of human interferon-beta protein.
3 Biotech, 8(1), 36.
R., Courtois, V., Peubez, I., & Mignon, C. (2012). Antibiotic-Free Selection for Bio-
Production: Moving Towards a New „Gold Standard “. Antibiotic Resistant Bacteria-
a Continuous Challenge in the New Millennium: InTech, 531-48.