Recombinant DNA Flashcards
Why are mRNA vaccines fast to develop?
mRNA technology allowed for quick adaptation once the virus’s genome was sequenced.
* Unlike traditional vaccines, they did not require virus culture or
protein purification.
How do you select a protein of interest?
Select a gene of interest that produces the desired protein.
* The gene of interest can be chemically synthesized but we first
need to know its coding sequence.
* We use complementary DNA (cDNA) libraries to collect the
desired gene’s DNA sequence
How is mRNA isolated from the cell?
Since the gene is expressed as
RNA, it can be reverse-transcribed
into cDNA.
* First, isolate mRNA from cells
expressing the target gene.
* Then use reverse transcriptase to
convert mRNA into single-stranded
cDNA.
* How? mRNA has a poly-A tail
* A short oligo (dT) is annealed to
this tail to serve as primer for
initiating DNA synthesis by the
enzyme reverse-transcriptase (5’-
3’).
* Reverse-transcriptase uses mRNA
as a template to synthesize a
complementary DNA strand.
* Next, the DNA polymerase enzyme is
used to synthesize a second DNA
strand.
* The outcome: a double-stranded
cDNA. Linkers or adaptors are added
to the cDNA ends to facilitate
cloning or sequencing.
* Next, sequencing methods are
employed to determine the cDNA
sequence.
* A cDNA library is a collection of
complementary DNA (cDNA)
molecules that represent the
expressed genes in a specific cell,
tissue, or organism at a particular
time.
* The gene of interest that codes for a
specific protein can now be
synthesized
How is the transfer vector prepared?
The next step is selection of a transfer vector.
* Vector is a DNA molecule utilized as a carrier to
transport a specific DNA segment into a host cell
as part of cloning.
* Vectors can take various forms:
* Plasmids (up to 10 kb)
* Cosmids (up to 30 kb)
* Viral vectors (35 kb or more)
* Artificial chromossomes (100 kb or more)
* Selection of a vector depends on the size of the
insert DNA.
* Plasmids are the most commonly used. They can
be found in many organisms- including Bacteria.
* After the preparation of the vector the next step in
the cloning procedure is restriction enzyme
digestion of gene selected and vector.
Restriction enzyme digestion
*DNA can be cut in specific sites using
particular restriction enzymes (restriction
endonucleases).
* Many restriction enzymes have been
identified from various bacterial strains.
* Each restriction enzyme recognizes a
specific DNA binding site.
* Restriction enzymes hydrolize the
covalent bonds of the phosphodiester
bond.
What do restriction endonucleases do?
*These enzymes can cleave DNA at specific sites-
pallindromic.
* Resulting in the creation of sticky ends (here
HindIII).
* Others cleave DNA at specific sites, producing
blunt ends (here SmaI).
How are DNA molecules ligated?
*The solution containing our treated gene is
added to a new tube.
* Afterward, the solution with the treated
plasmid vector is also added to the new tube
* For the ligation process DNA ligase enzyme is
added.
* The gene is now integrated into the vector
How does the bacteria become transformed?
*The recombinant vector is introduced into a
host cell, commonly E. coli, yeast, insect, or
mammalian cells It is called transformation
(prokaryotes) or transfection (eukaryotes).
* There are several transformation protocols. In
chemical bacterial transformation CaCl2 is
employed.
* Competent cells (e.g. E Coli) serve as host
cells.
* CaCl2 is added to a new tube on ice, followed
by competent cells, and finally the mixture of
recombinant DNA.
* CaCl2 causes fixtures in the cell membrane
allowing the plasmid to slip in.
What facilitates the binding of the plasmid to the bacterial cell surface?
Ca2+
What is the purpose of heat shocking the sample?
*To perform the heat shock the
tube is removed from the ice
and is then incubated at 42 C for
90s.
* It creates temporary fixtures on
the cell surface and allows for
plasmids to enter the cells.
* The tube is placed back on ice
for 2 minutes. Cells recover.
* A recovering medium (with
aminoacids, sugars etc) is
added to the bacterial cells for
their full recovery. Incubated
again at 37 C.
How is the recombinant DNA grown?
Using a bioreactor
How is the recombinant protein purified?
The recombinant protein is extracted from
the host cells and purified using
chromatography techniques (e.g., affinity,
ion exchange, size exclusion).
* The first step includes cell membrane
disruption: lyasis.
* After centrifuging the tube, there’ll be a
pellet at the bottom.
* Adding lyasis solution will disrupt the
bacterial cell membrane.
* Once the cell content is exposed, affinity
chromatography can be employed.
* Our desired protein will bind to the
stationary phase while others will pass
through.
* Following this- washing, and finally
elution take place.
* The elution buffer disrupts the specific
binding interactions keeping the protein
bound to the stationary phase.
* Finally, our desired protein is ready for
use.
What are the main steps in PCR?
- Preparation of reaction mixture.
* Template DNA (containing the target sequence).
* Primers (short DNA sequences complementary to target
DNA).
* DNA polymerase (e.g., Taq polymerase).
* dNTPs (deoxynucleotide triphosphates - A, T, G, C).
* Buffer solution (maintains optimal reaction conditions). - Denaturation (94–98°C). Heat to separate the DNA
into single strands. - Annealing (50–65°C). Cool to allow primers to bind to
their complementary sequences. - Extension/Elongation (72°C). DNA polymerase
extends the primers by adding complementary
nucleotides to synthesize new DNA strands. - Repeat cycles. Steps 2–4 are repeated for 25–40
cycles, doubling the amount of target DNA with each
cycle.
What is RT-PCR used for?
- diagnostic laboratories for identification of infections.
- diagnostic laboratories for identification of RNA based pathogens in plants. Assists in disease surveillance.
- Disease biomarker detection.
-Gene expression
What hormones can be produced with recombinant DNA?
-insulin
-human growth hormone
What vaccines are produced by recombinant DNA?
- Covid-19 vaccines
-HPV vaccine
-Hepatitis B vaccine
