Biotechnology

What is Gene Cloning?

• Also referred to as complementary DNA (cDNA) cloning or
cloning of DNA fragments obtained from cDNA.
• It involves the copying of mRNA transcripts into DNA
• DNA fragments are then inserted into bacterial plasmids
and then placed into bacteria or other expression systems
by transformation.

First strand cDNA synthesis:

• The synthesis of DNA from an RNA template, via reverse
General Steps in cDNA or Gene Cloning transcription, results in complementary DNA (cDNA).
▪ Extraction of total RNA • Total RNA is routinely used in cDNA synthesis for
▪ First strand cDNA synthesis downstream applications such as RT-(q)PCR, whereas
▪ RT-PCR specific types of RNAs (e.g., mRNA and small RNAs
▪ Purification of DNA fragments (PCR products) (miRNA) may be enriched for making cDNA library and
▪ Ligation into plasmid miRNA profiling.
▪ Transformation into bacterial expression system • Maintaining RNA integrity is critical and requires special
▪ Extraction of plasmid from bacterial cells precautions from extraction to experimental use (wearing
▪ Nucleotide sequencing gloves, pipetting with aerosol-barrier tips, using nuclease-
free lab-ware and reagents, and decontamination of work
Extraction of total RNA areas.
Isolation of mRNA: • Optimal purification methods remove endogenous
• A crude extract of the tissue with the gene of interest is compounds, like complex polysaccharides and humic acid
prepared. The extract must be free from proteins, from plant tissues that interfere with enzyme activity; and
polysaccharides and all other contaminants. common inhibitors of reverse transcriptases, such as salts,
• The technique of oligo-deoxythymine (oligo-dT) cellulose metal ions, ethanol, and phenol. Once purified, RNA should
chromatography is used for further purification of many be stored at –80°C with minimal freeze-thaw cycles.
eukaryotic mRNAs from the total polysomal fraction.
• mRNAs consist of poly A (adenosine residues) tail at their
3’ end. Under favorable conditions, this tail will bind to a
string of thymidine residues immobilized on cellulose and
then the poly (A)+ fraction can be eluted.
• To confirm if the extracted mRNA consists of the sequence
of interest, translation of mRNA in vitro and identification of
suitable polypeptides in the products obtained need to be
done.
• Trace amounts of genomic DNA (gDNA) may be co-purified
with RNA which can interfere with reverse transcription and
may lead to false positives, higher background, or lower
detection in sensitive applications such as RT-qPCR.
• The traditional method of gDNA removal is the addition of
DNase I to preparations of isolated RNA. DNase I must be
removed prior to cDNA synthesis since any residual
enzyme would degrade single-stranded DNA.
Unfortunately, RNA loss or damage can occur during
DNase I inactivation treatment.
• As an alternative to DNase I, double-strand–specific
DNases can be used to eliminate gDNA without affecting
 RNA or single-stranded DNAs. Their thermolabile property
allows simple inactivation at lower temperature (e.g., 55°C)
without negative impacts and can be incubated with RNA
for 2 min at 37°C prior to reverse transcription to
streamline.

First strand cDNA synthesis:

Component Key Features

Maintaining RNA integrity is critical and
requires special precautions during
RNA template
extraction, processing, storage, and
experimental use.
Maintains a favorable pH and ionic
strength for the reaction. The supplied
Reaction buffer buffer may also contain additives to
• Most reverse transcriptases used in molecular biology are enhance the efficiency of reverse
derived from the pol gene of avian myeloblastosis virus transcription.
(AMV) or Moloney murine leukemia virus (MMLV). AMV Should be at 0.5-1 mM each,
reverse transcriptase possesses strong RNase H activity preferably at equimolar concentrations.
that degrades RNA in RNA:cDNA hybrids, resulting in dNTPs High-quality dNTPs, freshly diluted, are
shorter cDNA fragments (<5 kb). recommended to ensure proficient
• Although less thermostable, the MMLV reverse reverse transcription
transcriptase became a popular alternative due to its Reducing agent, often included for
monomeric structure. It has lower RNAse H activity making optimal enzyme activity. Reaction
it capable of synthesizing longer cDNA (<7 kb) at a higher efficiencies may be compromised if
DTT
efficiency. DTT or other additives precipitate;
• MMLV reverse transcriptase has been engineered for even hence, reaction components should be
lower RNase H activity (i.e., mutated RNase H domain, or dissolved and well mixed.
RNaseH–), higher thermostability (up to 55°C), and Often included in the reaction buffer or
enhanced processivity (65 times higher) resulting in RNase inhibitor added to the reverse transcription
increased cDNA length and yield, higher sensitivity, reaction to prevent RNA degradation.
improved resistance to inhibitors, and faster reaction times. Eliminate any RNases by using
nuclease-free water from a commercial
Table 1. Common reverse transcriptases and their attributes. source, DEPC (diethylpyrocarbonate)-
AMV MMLV ENGINEERED treated water.
Water
RT RT MMLV RT Contaminating RNases cannot be
RNase H activity High Medium Low removed by simple filtration, and
Reaction autoclaved water is not adequate
temperature because RNases are heat stable.
42º C 37º C 55º C
(highest
recommended)
Reaction time 60 Min 60 Min 10 Min • Reverse transcription reactions involves primer annealing,
Target length ≤ 5 Kb ≤ 7 Kb ≤ 12 Kb DNA polymerization, and enzyme deactivation. The
Relative yield (with temperature and duration of these steps vary by primer
challenging or Medium Low High choice, target RNA, and reverse transcriptase used.
suboptimal RNA)
• The critical step is during DNA polymerization. Reaction
temperature and duration may vary according to the primer
• The main reaction components for reverse transcription
choice and reverse transcriptase used. If using random
include primers, enzymes, RNA template (pre-treated to
hexamers, incubating the reverse transcription reaction at
remove genomic DNA), buffer, dNTPs, DTT, RNase
room temperature (~25 °C) for 10 min after enzyme
inhibitor, and RNase-free water.
addition to extend the primers is recommended.
• A thermostable reverse transcriptase allows a higher
reaction temperature (e.g., 50°C) to help denature RNA
with high GC content or secondary structures without
impacting enzyme activity. High temperature incubation
 can result in an increase in cDNA yield, length, and possible of around 100º C, which
representation. complementarity with are usually sufficient to
• Polymerization time depends on a reverse transcriptase’s both ends of the denature most proteins.
sequence of interest that Thermus aquaticus find
processivity, or the number of nucleotides incorporated in
one wishes to amplify. its temperature of
a single binding event. Wild-type MMLV reverse
▪ One of the primers is comfort at 72º C,
transcriptase with low processivity often requires >60 min designed to recognize optimum temperature for
to synthesize cDNA; an engineered reverse transcriptase complementarily a the activity of its
with high processivity may take as little as 10 min to sequence located polymerase.
synthesize a 9 kb cDNA. upstream of the fragment
5’-3’’ strand DNA of
interest; the other to
RT-PCR:
recognize, always by
Steps in a Polymerase Chain Reaction (PCR) complementarity, a
• Denaturation or the separation of the two strands of DNA, sequence located
obtained by raising the temperature (around 94°C, called upstream
the denaturation temperature). The hydrogen bonds in complementary strand
DNA cannot be maintained at a temperature higher than (3’-5‘) of the same
fragment DNA.
80°C and the double-stranded DNA is denatured into
single-stranded DNA (single-stranded DNA).
Purification of DNA Fragments (PCR Products):
• Hybridization or annealing is carried out at a temperature
Restriction Enzymes
generally between 40 and 70°C, called primer hybridization
or annealing temperature. Decreasing the temperature
• Restriction enzymes also
allows the hydrogen bonds to reform and thus the
called restriction
complementary strands to hybridize. The primers, short
endonucleases or restrictase
single-strand sequences complementary to regions that
are enzymes that cleave DNA
flank the DNA to be amplified, hybridize more easily than
into fragments at or near
long strand template DNA. The higher the hybridization
specific recognition sites within
temperature, the more selective the hybridization, the more
molecules known as restriction
specific it is.
sites. These are produced by
• Elongation is carried out at a temperature of 72°C, called
bacteria and used to cleave
elongation temperature. It is the synthesis of the
foreign DNA, thus eliminating
complementary strand. At 72°C, Taq polymerase binds to
infecting organisms.
primed single-stranded DNAs and catalyzes replication
• A restriction enzyme is a
using the dNTPs present in the reaction mixture. The
protein isolated from bacteria that cleaves DNA sequences
regions of the template DNA downstream of the primers
at sequence specific sites, producing DNA fragments with
are thus selectively synthesized.
a known sequence at each end.
• It takes 20–40 cycles to synthesize an analyzable amount
• The use of restriction enzymes is critical to certain
of DNA (about 0.1 μg). Each cycle theoretically doubles the
laboratory methods, including recombinant DNA
amount of DNA present in the previous cycle.
technology and genetic engineering.
• It is recommended to add a final cycle of elongation at
• There are at least enzymes cuts a 3000 of them. Each one
72°C, especially when the sequence of interest is large
of these specific DNA sequence and doesn't discriminate
(greater than 1 kilo-base), at a rate of 2 minutes per kilo-
as to where the DNA comes from bacteria, fungi, mouse, or
base.
human, etc.
• PCR makes it possible to amplify sequences whose size is
• Restriction enzymes are one of the most important tools in
less than 6 kilo-bases. The PCR reaction is extremely rapid,
the recombinant DNA technology toolbox.
it lasts only a few hours (2–3 hours for a PCR of 30 cycles).
• Most restriction sites are 4 to 6 bases long, and most are
Primers Taq Polymerase
palindromic, meaning that the sequence reads the same
▪ To achieve selective ▪ DNA polymerase allows
amplification of replication. DNA forward and backward
nucleotide sequences polymerase purified or • HindIII, for example, is an H. influenzae restriction enzyme
from a DNA extract by cloned from of an that recognizes the sequence 5'AAGCTT-3' (upper strand)
PCR, it is essential to extremophilic bacterium, / 3'TTCGAA-5' (lower strand) and cleaves between the two
have at-least one pair of Thermus aquaticus, A's on both strands. (Here, the upper and lower strand
oligonucleotides. These which lives in hot springs sequences are the same but reversed.)
oligonucleotides will and resists temperatures
• The first three letters of a restriction enzyme’s name are
serve as primers for above 100º C is used.
replication, are This polymerase (Taq abbreviations of the bacterial species from which the
synthesized chemically polymerase) can enzyme has been isolated (e.g., Eco- for E.coli and Hin- for
and must be the best withstand temperatures H. influenza), and the fourth letter represents the particular
 bacterial strain. Roman numerals are also used as part of
the name when more than one restriction enzyme has been
isolated from the same bacterial strand.
EcoRI
HindIII

Ligation into Plasmid

Categories of Restriction Enzymes

Type I – which recognize specific DNA sequences but make their
cut at seemingly random sites that can be as far as 1,000 base
pairs away from the recognition site. Examples are EcoB and
EcoK.
Type II – which recognize and cut directly within the recognition
site. Examples are HindII and HindIII.
Type III – which recognize specific sequences but make their cut
at a different specific location that is usually within about 25 base
pairs of the recognition site.

• Ligation is the final step in the construction of a

recombinant plasmid that connects the insert DNA (gene
or fragment of interest) into a compatibly digested vector
backbone.
• This accomplished by covalently connecting the sugar
backbone of the two DNA fragments by the T4 DNA ligase
enzyme. The DNA ligase catalyzes the formation of
covalent phosphodiester linkages, which permanently join Transformation into Bacterial Expression System
the nucleotides together. After ligation, the insert DNA is
Different Expression Systems
physically attached to the backbone and the complete
plasmid can be transformed into bacterial cells for
propagation.
• The majority of ligation reactions involve DNA fragments
that have been generated by restriction enzyme digestion.
Most restriction enzymes digest DNA asymmetrically
across their recognition sequence, which results in a single
stranded overhang on the digested end of the DNA
fragment. The overhangs, called "sticky ends", are what
allow the vector and insert to bind to each other. When the
sticky ends are compatible, meaning that the overhanging
base pairs on the vector and insert are complementary, the
two pieces of DNA connect and ultimately are fused by the
ligation reaction.
Advantages of Yeast Expression System
• Carry out many post-translational modifications
(phosphorylation, glycosylation, and targeting).
• Readily grown in small and large scale bioreactors
• Secretes few proteins, the product can easily be purified.
• Generally recognized as safe (GRAS)
• Extensive screening of products is not recognized.

Insect Expression System

Pros:
• Produce proteins that has PTMs similar to mammalian
systems.
• Often properly folded and functional
• Ideal for producing moderate to high levels of
eukaryotic proteins for structure-function assays.
Cons: Nucleotide Sequencing
• Expensive
• Sometimes proteins are not correctly folded
• Often not stable
Cloning of Rabbitfish GH cDNA
Mammalian Expression System
Pros:
• Produce protein in the most native and active form.
• Have required PTM machinery to produce active and
useable protein used in mammals
Cons:
• Expensive
• Unstable
• Low yield and difficulties in purifying recombinant
proteins
• Limitations on the mechanisms of protein expression
Cloning the Full-Length cDNA
induction
• Extraction of Total RNA
• Almost always have over expression
• First strand cDNA synthesis
• Cloning the Internal Region
Extraction of Plasmid from Bacterial Cells
o Degenerate sense (5’ to 3’, 6) and anti-sense (3’
to 5’, 2) primers were synthesized based on the
most conserved regions of the amino acid
sequences of known teleost GHs and used for
PCR.

Checking for the presence of plasmids and inserts

- Select white colonies and culture
- Harvest bacteria and lyse to free plasmids
- Digest plasmids
- Run in agarose gel electrophoresis
* see figure 5
 number will be entered for you after you have specified a
sequence to design primers for,
Leave all the others ítems of this section as the default settings.

How To Design/Create PCR Primers Using Primer-BLAST

Primer-BLAST is NCBI’s free online primer design platform
which designs PCR primers using the Primer3 system and
simultaneously checks the likelihood that the primers will bind to
unspecific regions of the organism’s genome via the BLAST
algorithm.
1) Open the NCBI website. Go to the Nucleotide section
2) Search for your gene of interest. On the results page, use
the sidebars to filter the search further. Select ‘mRNA’ as
the ‘molecular types’ and the ‘Organism’.
3) Open up Primer BLAST.
Open Primer-BLAST to design PCR primers using the
sequence.
Select the ‘Pick Primers’ option on the right-hand sidebar
that has the heading ‘Analyze this sequence’.

Primer pair specificity checking parameters

The last section in Primer-BLAST contains a wealth of
parameters, most of which you can just leave alone. The most
4) Set the criteria for the desired primers. important part is to check that the ‘Organism’ box contains the
There are multiple sections to the Primer-BLAST page. correct name or organism number.
PCR Template Ensure the correct organism’s code/name is entered in the
The first section contains the accession number. The accession ‘Organism box’.
Leave all the other settings to their default values.

5) Run Primer BLAST.

Click the ‘Get Primers’ button at the bottom.
Primer-BLAST will now use the criteria you have specified to
design the PCR primers. This can take a few minutes depending
upon how conservative your design parameters are.
6) The output.
After running Primer-BLAST, the output window will be
displayed. The output can be split into two compartments: a
graphical view of the primer pairs and a detailed primer report.
In the example, 10 potential primer pairs were created based on
the parameters submitted to design the primers

7) Identifying the best primers END