RECOMBINANT

DNA
TECHNOLOGY
Prepared by: Ruem P. Tubo, LPT
 DEFINITION HISTORY ENZYMES & VECTORS
01 What is recombinant DNA 02 How did the rDNA 03 What are the enzymes
and rDNA technology? started? involved in rDNA? What are
vectors

PROCESS AMPLIFICATION
04 How does the rDNA 05 How to prepare multiple
technology works? copies of rDNA?
 TECHNIQUES APPLICATIONS
06 What are the techniques 07 Where can we apply the rDNA
identify a specific DNA, RNA technology?
and protein needed in rDNA
technology?

ETHICAL ISSUES RELATED STUDY

08 What are the ethical 09 Recombinant SARS-CoV-2 spike S1-
concerns raised when using Fc fusion protein induced high
rDNA technology? levels of neutralizing responses in
nonhuman primates
DEFINITION OF 01
RECOMBINANT
DNA What is recombinant DNA and rDNA
technology?
 molecules of DNA from two
different species that are inserted
into a host organism to produce new RECOMBINANT
genetic combinations that are of DNA
value to science, medicine,
agriculture, and industry.
.
RECOMBINANT
DNA
Also called as chimeric DNA.
Is one of the term (along with genetic RECOMBINANT
engineering, genetic modification,
and gene splicing) used for the direct
DNA
manipulation of an organisms gene. TECHNOLOGY
 02
HISTORY
How did the Recombinant
DNA started?
 Hamilton Smith, isolates the
first restriction enzyme (an
enzyme that cuts DNA at a very 1970
specific nucleotide sequence.)

HISTORY OF Stanley Cohen and Herbert

rDNA 1972 Boyer combine their efforts to

create recombinant DNA.

Technology Somastotatin which regulates

human growth hormones is
the first human protein made 1978
using recombinant technology
ENZYMES AND
VECTORS
03
What are the enzymes
involved in rDNA? What
are vectors?
 Enzymes in r-DNA Technology

Restriction Cleaves DNA at a specific

Endonuclease base sequence

DNA Ligase Binds down two DNA

molecules or fragments

DNA Fills single- stranded gaps in

duplex DNA by stepwise
Polymerase addition of nucleotides to 3’
ends (remove RNA polymer)
 Enzymes in r-DNA Technology

Reverse Makes a DNA polymer of an

Transcriptase RNA polymer

Adds a phosphate to the 5’

Polynucleotide OH end of a polynucleotide,
Kinase to label it or permit
ligation

Alkaline Removes terminal phosphates

Phosphatase from the 5’ end, the 3’ end or
both.
Restriction Endonuclease

• discovered by Arber,
Smith and Nathans in
the early 1970’s
• Found in bacteria
• Protect the bacteria
against viral infections
Restriction Endonuclease

They are named after the bacterium in which they are found.
Examples:
• Hin I------------- Haemophilus influenzae
• Hae III ---------- Haemophilus aegyptius
• Eco RI -----------Escherichia coli
 Restriction sites and fragments
The site recognized Restriction DNA fragments
Restriction site produced by
by a restriction fragments restriction
enzyme
enzymes
 Restriction Endonuclease

• Each restriction enzyme recognizes a specific base sequence in

double-stranded DNA and splits the DNA at this site.
• The base sequence recognized by a restriction enzyme is 4-8 base
pairs long which is a palindromic.
 Palindromic Sequence

• In DNA, the base sequences are read in 5’ -> 3’ direction. If a

sequence reads the same on both the strands in 5’ -> 3’
direction, it is known as a palindromic sequence
 Blunt VS Sticky
are also known as are also known as
Sticky ends cohesive ends or
Blunt ends even ends or non-
overlapping ends overlapping ends
Sticky Ends

They can easily be ligated to the complementary

sticky ends of another fragment of DNA by DNA ligase
A vector is required to transfer
Vectors
recombinant DNA into the cells.
 Vectors

are small circular double-stranded DNA

Plasmids molecules. Plasmids can accept foreign
DNA fragments up to 10 kb (kilo bases) in
size.
are viruses that infect bacteria. These
viruses have a DNA genome
Bacteriophages surrounded by a protein coat. DNA
fragments up to 20 kb in size can be
inserted in phage vectors, and can be
cloned in E.coli.
are hybrids of plasmids and lambda
Cosmids phage. DNA fragments up to 45 kb in
size can be inserted in cosmids.
 Vectors
Bacterial Artificial
Chromosomes (BAC)

is a genetically engineer vector. It is constructed

from a fertility plasmid ( F factor). It can accept
foreign DNA of 100-300 kb

Yeast Artificial
Chromosomes (YAC)
are formed by adding yeast telomeres to a
centromere – containing plasmid. Foreign
DNA up to 3000 kb in size can be inserted
in YAC, and cloned in yeast cells.
PROCESSES
04
How does the rDNA
technology works?
 Recombinant DNA Technology Process

Insertion of the Amplification of the

Isolation of the gene to the vector recombinant DNA
gene of interest molecule and molecule in the host
ligation cell

Preparation of
vector DNA and Introduction of the
DNA to be cloned vector DNA to the
appropriate host cell

This “sticky ends” from two
different DNA molecules
can hybridize together; then
the nicks are sealed using
ligase
1 2
The result is
recombinant
DNA
When this recombinant vector
is inserted into E. coli, the cell
processes the instructions and
by translation and transcription,
it assembles the amino acids
forming the protein product of
interest
3 4
More importantly, the
new instructions are
passed along to the next
generation of E.coli cells
forming recombinant
clones on the culture
agar
Amplification
05
How to prepare multiple
copies of rDNA?
 AMPLIFICATIONS
Cloning of DNA

Polymerase Chain Reaction (PCR)

CLONING of DNA

The term clone means exact

copy of the parent. A
duplicate or a look alike
carrying the same genetic
signature or genetic map
CLONING of DNA
using Plasmid
vector
CLONING of
rDNA Plasmid
CLONING of
rDNA
Bacteriophages
CLONING of
rDNA Cosmids
CLONING of
rDNA BACs
CLONING of
rDNA YACs
Is a technique for rapid amplification Polymerase
of DNA devised by Kary Mullis in the Chain Reaction
1980s. The only limitation of PCR is
the size of DNA that can be amplified. (PCR)
Thermus Aquaticus

• is a bacterium
found in hot water
springs.
• The DNA
polymerase of this
is used for PCR
Polymerase Chain Reaction (PCR)
 Strand
3 Steps of PCR separation
(Denaturation)
Primer binding
(Annealing)

Amplification
Primer extension
(addition of
deoxyribonucleotides
to primer)
Polymerase Chain Reaction (PCR)
Thermocyclers

Strand
Primer binding
separation
(Annealing)
(Denaturation)

Primer extension
(addition of
deoxyribonucleotides to
primer)
 Several new variants of the
classical PCR have been devised:
Polymerase
Chain Reaction • Nested PCR
(PCR) • Multiplex PCR
• Reverse transcriptase- PCR
• Quantitative PCR
Techniques
05
What are the techniques identify
a specific DNA, RNA and protein
needed in rDNA technology?
 Probes

Complementary
DNA (cDNA) Are used for identification of DNA and RNA
probes

Can be used as probes to identify Antibodies

specific proteins
 Techniques in DNA identification

Southern blotting
 Techniques in RNA identification

Northern blotting
 Techniques in Protein identification

Western blotting
 Techniquesfor determining base
sequence of DNA

Chemical method of
Maxam- Gilbert
Techniquesfor determining base
sequence of DNA

Enzymatic dideoxy
method of Sanger
Applications
of rDNA
06
technology Where can we apply the rDNA
technology?
 • Pharmaceutical and therapeutic applications
• Insulin for diabetes
• Factor VIII for males suffering from
hemophilia A.
• Factor IX for hemophilia B
Applications of • Erythropoietin for treating anemia
• Human growth hormone
recombinant • Several types of interferon
• Granulocycle-macrophage colony-
DNA •
stimulating factor (GSM-CSF)
Many monoclonal antibodies
technology • Gene therapy
• medical diagnosis
• Xenotransplants
• Food products
• Better Crops (drought & heat resistance)
• Plants that produce their own insecticides
ETHICAL
ISSUES
07
What are the ethical concerns raised
when using rDNA technology?
Commercialized and become
big source of income for
businessman Effects natural immune
system of the body

Can destroy natural

ecosystem that relies on
Major international concern:
organic cycle.
manufacturing of biological
weapons such as botulism &
anthrax to target humans with
specific genotypes

Prone to cause mutation that

could have harmful effects

Concerns of creating
super- human race
Related Study
08
Recombinant SARS-CoV-2 spike S1-Fc fusion
protein induced high levels of neutralizing
responses in nonhuman primates
What was the focus of the study?

The current study in non-human

primates shows that the use of
the recombinant spike fusion
protein, S1-Fc, on the virus,
prevents infection by inhibiting
the fusion of the virus to the host
cell membrane.
How was the vaccine synthesized?

The researchers fused the full length SARS-CoV-2 S1

protein with the Fc region of human IgG1 as our
vaccine candidate and expressed the recombinant
protein using a stable CHO-K1 cell line. They
purified the resulting protein and added an
adjuvant to increase its immunogenicity. This was
then tested in mice, rabbits, and monkeys, and the
antibodies formed were analyzed..
 Why is this study important?
The most significant advantages of this approach
are the reduced costs of production and the ease of
approach.
The possible risks of this approach are dealt with by
the authors. For instance, multiple doses were
required to produce a stable and robust antibody
response. This is amenable to the same type of
immunization schedule required by many other
vaccines, including the vaccine against shingles.
 Why is this study important?
Secondly, the paper claims that using a single 3,000 L
CHO cell reactor (the CHO-K1 line) was used to
generate the antibodies), 3 million doses of the
recombinant S1-Fc fusion protein vaccine can be
produced within two weeks. They say, "With so many
antibody drug production plants in the world, it may
be the ONLY feasible way to make enough COVID-19
vaccines for the entire world within one year."
 Why is this study important?
Thirdly, they point out that many biologicals already in use
contain Fc components derived from the host cell, without
additional side effects. Indeed, the presence of the Fc part
may help to mimic the 3D conformational structure of the
S oligomer in vivo, by the formation of disulfide bonds
between the Fc parts of two such molecules. Moreover,
and most significantly, it is easy to purify the recombinant
protein from the CHO-K1 cell when the S1 protein is fused
to the human Fc domain, following the standardized
antibody-drug protocols.
 Materials of the study

• AD11.10 (saponin based microemulsion)

• AD20Gold+ (nano emulsion with synthetic
MPL)
• Adjuvants were from Advaccine, China.
Freund's complete adjuvant (CFA) was
purchased from SIGMA, USA.
 Materials of the study
• Female BALB/c mice
were obtai n ed f r om
Vital River Co., China.
• New Zealand White
rabbits were purchased
and hosted at
Longan Co., China.
• Macaques were
generated and hosted
at Xieerxin Biotech.,
China.
 Methods of the Research

Pseudo virus
Immunization neutralization
assay

Enzyme-linked SARS-CoV-2
immune assay Neutralizing assay.
Methods of the study: Immunization
4-weeks old female BALB/c mice immunized with S1-Fc
protein immersed in AD20Gold+ (9.2μg on Day 0, 3, 7 and
reduced to 0.575μg on Day 9 and 11 intramuscularly).

NZW rabbits were also immunized with S1-Fc protein immersed in

AD20Gold+ (100μg on Day 0, 4, 7 and reduced to 50μg on Day 11, 14
and 18 intramuscularly).

For immunization of macaques, CFA was used to prime the

primate
at the first injection and AD11.10 was used to boost them (250μg
in CFA on Day 0 subcutaneously, and 250μg in AD11.10 on Day 4, 9,
22 and 26 intramuscularly).
 Enzyme-linked immune assay
(ELISA)
• 1.5 μg/mL of SARS-CoV-2 S1-6x Histidines protein in PBS, blocked with 3% BSA-PBS coated in
96-well plate
• Serial dilutions of sera were first diluted with the normal sera from the same species, further
diluted with Sample Dilution Buffer (20% Calf serum in PBS, 20%CS-PBS). 100μL of the diluted
samples were transferred to each well of the plates.
• The captured antibodies were probed with Horseradish-Peroxidase(HRP)-conjugated secondary
antibodies. For example, to detect monkey anti-S1 total antibodies, HRP-conjugated AffiniPure
Goat Anti-Human IgG (H+L) from Jackson Immuno Research was used.
• For IgM detection, HRP-labeled Monoclonal Mouse Anti-Human IgM (μ) from Cellway-lab was
used.
• After final washes, HRP substrate TMB solution (Beijing Kwinbon, China) was added and
absorbencies were measured at 450nm with a microplate reader.
 Pseudo neutralization assay
• HEK 293T cells and ACE2-overexpressed HEK 293T cells (ACE2-293T) were cultured in
DMEM (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum
(FBS, Gibco), 50 U/ml penicillin (Gibco) and 50μg/ml streptomycin (Gibco) at 37
degree C with 5% carbon dioxide.
• HEK 293T cells were co-transfected with 10μg of a plasmid encoding SARS-CoV-2 S
protein and 10μg of an env-deficient, luciferase-expressing HIV vector
(pNL4–3.luc.RE) using Lipofectamine 2000 reagents (Life Technologies, USA).
• After 48 h, pseudo virus-containing culture supernatants were collected,
filtered(0.45 μm), and stored at -80°C in 1ml aliquots.
• The 50% tissue culture infectious dose (TCID50) of a single thawed aliquot of
pseudo viruses was determined in ACE2-293T cells. For TCID50 measurement, serial
dilutions of pseudovviruses were added to ACE2-293T cells pre-seeded in 96-well
plates. After 12 h incubation, the medium was replaced by DMEM containing 10%
FBS, and cells were cultured for an additional 48 h.
 Pseudo neutralization assay
• ACE2-293T cells were seeded in 96-well plates at one day prior to infection. Serum
samples were inactivated at 56 degree C for 0.5h, and then 3-fold serially diluted and
incubated with 200 TCID50 of pseudoviruses for 1 h at 37°C.
• The mixtures were then used to infect ACE2-293T cells in triplicate. Following 12 h
infection, wells were replenished with fresh medium and the luciferase activities of
cells were determined 48 h later.
• Cells were lysed with lysis buffer (Glo-lysis buffer, Promega), followed by addition of
luciferase substrate (Bright-Glo luciferase assay substrate, Promega).
• Luciferase activity was measured using a GloMax 96 microplate luminometer
(Promega) and wells producing relative luminescence units (RLU) above three times
of the mean background value were defined as positive.
• The 50% neutralization titer was calculated by probit analysis using the SPSS
software.
 SARS-CoV-2 Neutralizing assay
• Strain CN1 was chosen for neutralizing assay.
• SARS-CoV-2 virus titer was determined by microdose cytopathogenic efficiency (CPE) assay. Serial 10-fold
dilutions of virus contained samples were mixed with 1-2 x104 Vero cells, and then plated in 96-well culture
plate.
• After 3-7 days culture in a 5% CO2 incubator at 36.5°C, cells were checked under a microscope for the
presence of Cytopathic effect (CPE).
• Virus titer was calculated by the method of Karber. Serum samples were inactivated at 56 degree C for 0.5h
and serially diluted with cell culture medium in two-fold steps.
• The diluted sera were mixed with SARS-CoV-2 suspension of 100 TCID50 in 96-well plates at a ratio of 1:1,
followed by 2 hours incubation at 36.5 degree C in a 5% CO2 incubator.
• 1-2 x104 Vero cells were then added to the serum-virus mixture, and the plates were incubated for 5 days at
36.5 degree C in a 5% CO2 incubator
• CPE of each well was recorded under microscopes, and the neutralizing titer was calculated by the dilution
number of 50% protective condition.
 What did the results show?

The subjects displayed a high concentration of neutralizing

antibodies by day 7. "On day 7, all of the immunized mice
showed syndrome of fevers, much earlier than usual, suggesting
S1-Fc fusion protein is super immunogenic," the researchers said.

On day 14, rabbits displayed strong neutralizing activity against

the virus when immunized with a pseudo virus neutralization
assay.
 What did the results show?

The investigators claim that their results show the S1-Fc protein is capable of
effectively stimulating the host to induce humoral immunity (antibodies), and
importantly, to produce neutralizing antibodies at high concentrations in a
primate species other than humans. This, they say, is a strong indication that
this recombinant fusion protein could be a strong candidate for COVID-19
vaccine development.

While the female monkey showed anti-S1 IgG antibodies only in the female
monkey on day 9, both monkeys developed strong titers of this isotype by day
16. Still, the IgM levels dropped below detectable concentrations.
 What did the results show?

An additional boost of the fusion protein on day 24 pushed up the

IgG levels and IgM levels, the former to a much larger extent. This
could throw light on the phenomenon in which both S1-IgM and
IgG were found to be present in patients with COVID-19. This could
be a sign of re-infection or that the dormant virus is re-emerging
from the infected cells.

In the recovered COVID-19 primate patients, IgG levels were

detected at four days from onset, to increase by day 12. On day 20
and 28, the levels dropped. IgM was not detectable at any time.
 What is the conclusion?

The study concludes, "Our data

strongly suggests that the CHO-
expressed SARS-CoV-2 S1-Fc
recombinant protein could be a strong
candidate for vaccine development
against COVID-19."
GENOMICS
Prepared by: Ruem P. Tubo, LPT
 DEFINITION GOALS HISTORY
01 What is genomics? How does 02 What are the goals 03 How did genomics
it differ with genetics? of genomics? started?

GENOME GENOME MAPPING

04 What is a genome? 05 How to locate the genes?
06 GENOME SEQUENCING 07 SUBFIELDS OF GENOMICS
How to sequence genome? What are the subfields of
Genomics

HUMAN GENOME APPLICATIONS

08 09
PROJECT Where can we apply genomics?

What is HGP? How did it

started?
 ETHICAL ISSUES
What are the ethical
10 concerns raised in genomics?

RELATED STUDY
11 Sequence analysis of SARS-
CoV-2 genome reveals
features important for
vaccine design
 01
DEFINITION OF
GENOMICS
What is genomics? How does it differ
with genetics?
Is the sub discipline of genetics devoted
to the mapping, sequencing and Genomics
functional analysis of genomes
 Genetics vs Genomics

Genetics Genomics

- Looks at a single
genes one at a - Looks at all genes
time in an organism

- involves the study - addresses all

of functions and genes and their
the composition of interrelationships
the single gene
 02
GOALS OF
GENOMICS
What are the goals of genomics?
 • Sequence the entire genome by cutting it into small,
manageable pieces
• Assemble the entire genome from the pieces
• Understand how gene expression takes place
• Compile the genomic sequences of organism
• Search out the location of the genes for analyzing
Goals spatial relationships and annotate the gene set in a
genome
• Learn the function of genes and their influence
• Establish how gene expression profiles of a cell
vary under different conditions
• Compare gene and protein profiles among different
organism to learn about evolutionary relationships
HISTORY OF
GENOMICS
03
How did genomics started?
 Genomics is a concept that was
first developed by Frederick
Sanger, who first sequenced 1970s
the complete genome of a
virus and of a mitochondrion

HISTORY OF Walter Fiers and his research

group became the first to

GENOMICS 1972 sequence a gene. They

sequenced the gene of
Bacteriophage MS2

Hamilton Smith and his team

became the first to sequence a
genome of a free living 1995
organism- that of Haemophilus
influenzae
GENOME
04
What is a genome?
The whole hereditary information of an
organism that is encoded in the DNA.
Genome
Genome
 • The human genome is by far the most
complex and largest genome
• Its size spans a length of about 6 feet
of DNA containing 30, 000 to 40, 000
genes.
• The DNA material is organized into a
Human haploid chromosomal set of 22 and a
sex chromosome.
Genome
G

T
 Introns vs. Exons
Introns Exons

derived from the term “intragenic are coding sections that

regions”, are non- coding sections remain in the mRNA
and does not remain in mRNA sequence
Gene
expression
GENOME
MAPPING
05
How to locate the genes?
Genome we can identify the location
of the gene and the
Mapping distance between the genes
 • The term marker is used very broadly
Genomic to describe any observable variation
that results from an alteration, or
mutation, at a single genetic locus.
Markers
• Acts as signs or flags
 Linkage
mapping

Genome
Mapping
Restriction
mapping

Physical FISH
Mapping mapping

STS
mapping
 we can determine the probability of
Linkage the genes whether they are near or
distant with each other in a
Mapping chromosome but not the exact
location
Linked vs Unlinked genes
 • a restriction map is constructed
Restriction by cleaving DNA into fragments
and measuring the distances
Map between the sides of cleavage
12 bp
12 bp
12 bp
12 bp
12 bp
12 bp
 • Fluorescence in situ
Hybridization
FISH • Used for finding specific
features of the DNA which is
mapping used in medicine and also help
to detect and determine the
location of the gene
FISH
mapping
 - Sequence tagged site
- is simply, generally between 100-
STS a short DNA sequence500 bp in
length, that is easily recognizable
Mapping and occurs only once in the
chromosome or genome being
studied.
STS
Mapping
 GENOME
SEQUENCING
06
How to sequence genome
 • Genome sequencing or DNA
sequencing is figuring out the
Genome order of DNA nucleotides, or
bases, in a genome—the order of
sequence As, Cs, Gs, and Ts that make up
an organism's DNA.
 Genome Sequencing Approaches
Hierarchical Shotgun
sequencing
useful for sequencing genomes of
higher vertebrates that contain
repetitive sequences

Whole genome shotgun

sequencing-

useful for smaller genomes

SUBFIELDS OF
GENOMICS
07
What are the subfields of Genomics
 Structural
Genomics

Genetics Genomics Functional

Genomics

Comparative
Genomics
 Subfields of Genomics

Structural aims to determine structure of every

protein encoded by the genome

aims to collect and use data from

Functional sequencing for describing gene and
protein function.

Comparative aims to compare genomic features

between different species
 HUMAN
GENOME
08
PROJECT What is HGP? How did it started?
Human • The US Human Genome Project is a 13 year effort
which is coordinated by the Department of Energy

Genome •
(DOE) and National Institute of Health ( NIH)
This project was launched in 1986 by Charles DeLisi
and was originally planned to last for 15 years
Project
 • Identify the approximate genes in human DNA
• Determine the sequences of 3 million chemical
base pairs that make up human DNA
Goals of •
•
Store this information in databases
Improve tools for data analysis
•
HGP Transfer related technologies to the private
sector
• Address the ethical, legal, and social issues
(ELSI) that may arise from the project.
 History of Human Genome Project

Project initiated
Welcome Trust
as joint effort of
joins the project
US DOE and NIH

1986 1990 1994 1996 1998

The birth of Genetic Privacy Act: to Celera Genomics

Human Genome regulate collection, formed to sequence
Project analysis, storage, and much of the human
use of DNA samples genome in 3 years
and genetic
information is
proposed.
 History of Human Genome Project
HGP sequencing is
completed and
Completion of the project is
working draft of declared finished
the entire human 2 years ahead of
genome schedule

1999 2000 2001 2003

Completion of the Analysis of the working

sequence of draft are published
chromosomes 22- the
first human
chromosome to be
sequenced
• used samples from a blood ( female)
and sperm (male) from a large number
of people. Whose
• Celera Genomics collected samples Genome is
from individuals who were Hispanic,
Asian, Caucasian, and African- sequenced?
American.
APPLICATION
09
Where can we apply genomics?
 • It can be used in the field of medicine for
early detection of genetic disease and its
diagnosis and treatment
• It is also useful in agriculture livestock
breeding and bioprocessing.
• To study evolution through mutation lineages
• In forensic science
Benefits • Identity comparison for nucleic acid
sequences
• Analysis of gene expression profile
• Database of model organism
• Hunting for disease -related genes
• Analysis of the genes related to drug action
• Screening of poisonous side effect genes.
ETHICAL
ISSUES
10
What are the ethical concerns raised
in genomics?
Privacy and
confidentiality issues Benefit sharing

Informed consent Public data release

Withdrawal from research Commercialization

Genetic discrimination
RELATED
STUDY
11
“Sequence analysis of SARS-CoV-2
genome reveals features important
for vaccine design”
 What is the study all about?
The study is all about a promising approach for
vaccine development and that is to generate an
attenuated virus through codon pair deoptimization.
This approach carries the advantage that it only
requires limited knowledge specific to the virus in
question, other than its genome sequence.
Therefore, it is well suited for emerging viruses, for
which we may not have extensive data. .
 Why is this study important?
This study will lead to better understanding of the
factors that make codon pair deoptimization
successful in generating attenuated viruses.
Furthermore, this will provide the scientific
community with the framework that would allow
anyone to independently proceed towards
engineering a codon deoptimized SARS-CoV-2 for
vaccine development.
 Methods of the Research

Sequence accession
and codon comparison RNA Folding

Comparison of
Examine viral gene
Codon and codon
codon usage
pair usage in
properties
species
 Materials of the Research
For Sequence accession and codon comparison:

1. The complete reference sequences for severe acute respiratory syndrome

coronavirus 2 (SARS-CoV-2 , accession NC_045512.2) were downloaded
from NCBI RefSeq49 on June 8, 2020.
2. CDS sequences from 5,064 complete SARS-CoV-2 isolates were
downloaded using NCBI SARS-CoV-2 data hub on June 8, 2020.
3. Sequences of poor quality or with CDS lengths that did not match those of
the reference sequence due to deletion or insertion were removed. To
calculate percent difference in codon usage, each CDS was compared at
the codon level to that of the reference sequence. Codons containing
nucleotides where a base call could not be made (“N”) were removed from
the calculation. All scripts for this calculation were written in Python 3.7.4.
 Materials of the Research
For Comparison of Codon and codon pair usage in species

1. Codon, codon pair and dinucleotide usage data for Homo sapiens, Canis
lupus familiaris, Chiroptera (bats) and Pholidota (pangolins) were
downloaded from the CoCoPUTs database5 on March 13, 2020.
2. Likewise, human lung, kidney (cortex) and small intestine (terminal ileum)
tissue-specific codon, codon pair and dinucleotide usage data were
accessed from the TissueCoCoPUTs database28 on March 13, 2020.
3. Codon, codon pair and dinucleotide usage data for SARS-CoV-2 was
calculated from the reference sequence (accession NC_045512.2) using
scripts written in Python 3.7.4. Euclidean distances between codon pair
usage frequencies were calculated using the dist function from the stats
package in R 3.6.1.
 Materials of the Research
For the RNA folding:

1. NuPack to predict the minimum free energy (MFE) secondary structure on the
100 nucleotides following the frameshift35,
2. ,LandscapeFold, to examine the full structure landscape of the 75 nucleotides
following the frameshift36.
3. MatLab’s Needleman-Wunsch sequence was used for sequence alignment
implemented on the 100 nucleotides following the frameshift using default
parameters. The parameters employed were the defaults: the NUC44 scoring
matrix and a gap penalty of 8 for all gaps.
4. Structure alignment was measured using a method similar to the researchers
previously-studied “per-base topology” score36.
 Materials of the Research

For examining viral gene codon usage properties:

1. RSCU (Relative synonymous codon usage) was calculated as defined in Sharp et

al.38 based on Homo sapiens genomic codon usage data accessed from the
CoCoPUTs database5 on March 13, 2020.
2. CPSs (codon pair scores) for all 4,096 codon pairs were calculated as described in
Coleman et al.2 using Homo sapiens genomic codon pair usage data accessed from
the CoCoPUTs database5 on March 13, 2020.
3. CPB (codon pair bias) of a gene is the arithmetic mean of all CPSs throughout the
gene, as defined in Coleman et al.2.
 What was the result of the study?
The result of the research pointed to the S and N genes
as potential targets for codon pair deoptimization. The S protein,
which binds to the cell-surface receptor and induces virus-cell
membrane fusion, has the highest CPB score of all viral proteins,
leading to significant flexibility for codon pair deoptimization.
The N protein, which forms the ribonucleoprotein
complex with the virus RNA, is the most conserved and stable
protein among the coronavirus structural proteins. It uses
mostly codon pairs with intermediate frequency; thus, it could
be substantially codon pair deoptimized.
 What was the conclusion of the study?

So the analysis identified the spike (S) and nucleocapsid (N)

proteins as promising targets for deoptimization and
suggests a roadmap for SARS-CoV-2 vaccine development,
which can be generalizable to other viruses.
 RESOURCES
RECOMBINANT DNA TECHNOLOGY

q https://www.britannica.com/science/recombinant-DNA-technology/Genomics

q https://www.britannica.com/topic/philosophy-of-biology/Social-and-ethical-issues#ref1115319

q https://www.britannica.com/science/recombinant-DNA-technology/Genomics

q https://www.powershow.com/view0/8d70b8-ZTQ2O/Recombinant_DNA_Technology_-
_Tools_and_Techniques_powerpoint_ppt_presentation?varnishcache=1

q https://www.slideshare.net/seetaram443/recombinant-dna-rdna-technology

q https://www.news-medical.net/news/20200426/Recombinant-protein-produces-neutralizing-COVID-19-
antibodies-in-primate-model.aspx
 RESOURCES
GENOMICS
q https://www.slideshare.net/pubudu/genomic

q https://www.slideshare.net/queenmalik/genomics-58002262

q https://www.slideshare.net/khushbo0/types-of-genomics-ppt

q https://www.slideshare.net/krajgire43/genomics-66766372

q https://www.nature.com/articles/s41598-020-72533-2#data-availability
 Any
questions?
