Recombinant DNA Technology: Procedure and Process

This study material from Desklib provides an overview of Recombinant DNA Technology, including the use of PCR, agarose gel preparation, and various enzymes in DNA technology. It discusses the procedure and process of rDNA technology, selection of perfect vector, and the properties required in PCR technique. The study also covers the results obtained from the experiment and the discussion on the technique of rDNA.

GENETICS

Table of Contents
ABSTRACT.....................................................................................................................................1
INTRODUCTION...........................................................................................................................2
MATERIAL AND METHOD.........................................................................................................3
Safety instruction in lab session.............................................................................................3
PCR reaction Mixture.............................................................................................................3
Step to prepare agarose gel.....................................................................................................4
RESULTS........................................................................................................................................5
DISCUSSION..................................................................................................................................7
CONCLUSION................................................................................................................................8
REFERENCES................................................................................................................................9

ABSTRACT
The experiments are completely based on the plasmid vector which us used in the molecular
cloning experiment where a double stranded molecule of the DNA which help to replicate within
the bacterial cell. In this, the number of plasmid is used on the DNA technology which is
associated with the recombination. They usually configure different properties which help to
provide information which include origin of replication which usually refers the capability which
must replicate in the bacterial host. As per this, the genetic marker is used which have properties
of antibiotic resistance marker and some cloning vector plasmid which have two markers.
Usually, the summary form the lab session includes the recombinant DNA aspect on the practical
level which include different and compound which configure various reaction channel which
trigger the experiment in the lab. The use of polymerase chain reaction with the amplification
with the cDNA inserts from the three recombinant DNA plasmid.
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INTRODUCTION
Genetics is defined as the study which is based on the heredity in the general way and
associated with the gene in the particular way. Moreover, the genetics is defined as the form of
central level of pillar which is related chapter of biology. It also shows some of overlap with
number of areas which include agriculture, medicine and biotechnology. However, with the
knowledge of depth, it is also evaluated that it is also a study which defined different qualities
called traits are passed from parents to their children (Beltrán‐Corbellini and et. al., 2020). The
genetics help to create understanding regards with the family such as what make unique and
different, why family member looks alike and why some disease run in families. The ways help
to get the information through genes. In this, research question includes concept of recombinant
DNA technology, selection of perfect vector includes plasmid or bacteriophage and whereas,
there are different set of compound and preparation which is essential to promote the reaction in
effective which show optimised result at the end, in this, other research question is include the
properties which is required in PCR technique and process to follow standard operating
procedure. In this, the main of this project to create better understanding regards with the rDNA
technology which is associated with procedure and process include agarose gel preparation and
polymerase chain reaction and other various enzyme which play role in DNA technology
(Kucirka and et. al., 2020).
The mechanism of the PCR indicate that a segment of DNA is using in the PCR, the sample
is usually heated so that the DNA denature or they get separated into the two piece of the single
strand DNA. As per this, the enzyme is also called Taq polymerase synthesis which used to build
two new strand of DNA, using the original strand as the template. In this, the major of process
which show the duplication of the original DNA. With each of the molecule they contain one old
and new strand of DNA. In addition, the denaturation and synthesis of the new DNA is usually
repeated as many of 30 to 40 times. As per this, the entire process of PCR which is automated
and can be completed in the just few hours. It is completely directed by the machine called
thermocyler. Restriction endonuclease are the bacterial enzyme which help to cleave double
stranded DNA. In this, the type I RE which is important in the function of bacteria which do not
cleave DNA at the specific sequences.
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RESULTS
With the aspect of results which is obtained from the experiment is create understanding
related with rDNA. In this, the Recombinant DNA named rDNA which is widely used in the
biotechnology, medicine and the research. Moreover, the protein and the other result which is
obtained from the result which is used for the rDNA technology that found essentially in the
pharmacy, doctor and use in any medical testing. There is retinoblastoma which defined as the
malignant tumour of the retina, neurofibromatosis which is defined as the presence which is
based on the multiple neurofibromas under the skin. Some of the recent advance which made the
possible by the recombinant DNA technology. The results indicate that they are isolating protein
in the large quantities which have number of product which is available and major include,
hormone named as follicle stimulating hormone, growth hormone, insulin and various associated
protein. In this, they are creating some of the possible mutation identification which is occur due
the technology which comes out with the people that they can easily tested for the protein which
configure with the mutation. Their presence shows breast cancer and so on (Pandey and et. al.,
2021).
Figure 1 Genetic mutation
(Source: Genetic mutation: https://www.nature.com/scitable/topicpage/genetic-mutation-1127/)
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With the contrast with the graph, it helps to show the overwhelming majority of mutation which
have small effect. In this, it is defined as the example which have best possible aspect of
distribution of the deleterious mutational effects which has been shown that it is obtained from
the sequence of DNA sequence polymorphism data from the natural population of the two
drosophila species. In this, the spike 10-10 include all smaller effects. As per this, the effect not
shown, if they include a structural change of damage that is equivalent towards the selection of
coefficient that is super lethal (Sefidi-Heris and et. al., 2020).
Figure 2 Results obtained from experiments
In this, the CCC monomer is repetitive and OC monomer is single which is linear in number. As
per this, the CCC dimmer show some raise but they are limited and OC dimmer have optimised
range include the outcome. This is enhanced due to the agarose gel.
DISCUSSION
The technique of rDNA shows various aspect over the insertion part of DNA that include
major aspect regards with the associated factor. With this, the diabolical structure, usually
discussion some part of application which configure with genetic engineering which is help to
completed by some vital step include three basic step which is isolation of DNA fragments form
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the donor organism. In the second one is used to indicate about the insertion of an isolated donor
DNA which have some fragments which include the vector genome, the last applied application
which is associated with the genetic engineering is determine as growth which is recombinant
vector in an appropriate host. Usually, they are involving with the six step which act as helpful
approach towards the rDNA technology (Zare and et. al., 2017).
These majorly include isolating genetic material, restriction enzyme digestion with the help
of using PCR for the amplification, ligation of the DNA molecule, and inserting for the
recombinant DNA into the host, and they focus on the isolation of recombinant cells form. The
PCR is very important aspect which is essential for the identification of organic factors which
include blood and other. In this, the different line shown the various factor which is enable the
PCR that have positive role in the crime determination and many more. As per this, the PCR
allow DNA that is determined from the tiny sample where a single molecule of the DNA is
enough for the PCR amplification. Whereas, restriction enzyme is most commonly used in the
molecular cloning technique such as PCR or the restriction cloning. In this, it is also used to
quickly check the identity of a plasmid by the diagnostic digest.
In this, the forwards option which is associated with the T7 primer which have 5’ named
TAA TACGA CTCAC TATG GG 3’ and the opposite reverse SP6 primer is 5’ named as
TTAGG TGACA CTATA GGA 3’
Whereas, the recombinant plasmid which was cleaved at the Ndel restriction site with the
help of Ndel restriction which is based on endonuclease enzyme for the single digest, moreover,
they are resulting in the single linear with the many base pairs. As per this, the Ndel and Hindlll
double used to digest the cleave which is based on recombinants plasmid at the respective that is
related with the Ndel and Hindlll recognition site which is found and moved towards the agarose
gel than the singles digest due to the smaller length. Usually, the digest cleaves the plasmid
which is help to produce the DNA into two shorter DNA fragments then the single digest which
formulate one long plasmid DNA. Moreover, the distance which is travelled by the DNA
fragment is completely depend on the size of fragments with the shorter fragment which is
moving towards the further. In this, the limitation towards the method which is in multiple
possible product that obtain as the part of results which is based on the DNA ligation and the
transformation of bacteria. In this, the wrong DNA inserts towards the plasmid vector. The bit
that show the wasteful parameter by the recombinant plasmid are usually obtained.
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The PCR primers anneals at the forwards T7 promotor and the reverse SP6 promotor by the
Taq DNA polymerase. The DNA polymerase which used to cause the 3’ end of the DNA
molecule that is associated with the double strand which have an extra adenosine. It also helps to
allow the DNA to ligate and should be cloned. However, the clone is usually amplified for the 25
to 35 times. The enhancing rate of the number of target which is based on the DNA doubles. The
PCR product is usually have some of the degree longer the endonuclease restriction that digest
anneal at the insert and this is usually seen in the PCR A and C in the agarose gel.
The method which is associated with polymerase chain reaction is useful aspect that is
helpful for the DNA fingerprinting but not produce in the aspect of cDNA. This is underlying
with the 3’ to 5’ by proofreading of the exonuclease activity based on the fingerprinting that
causes and do not responsible for the secreting of cDNA. There are some of limitation which can
be reduce by using the various DNA polymerase called the pyrococcus furiosus DNA
polymerase with the error which is noted as the average indicate the 1.3 * 10-6.
In this, the band is defined as the sample B for the both the PCR and double restriction
which help to digest that has same size which could be because they have isolated the form of
cDNA with the position. In the other words, the site of recognition of the double digest show the
T7 and SP6 act as promotor of the PCR are in the same position which show the result in the
similar number of base pairs.
CONCLUSION
As per the above discussion, it is analysed that the recombinant DNA technology play vital
role in the decline of disease which is based on genetic level. The recombinant DNA technology
is helpful in making of artificial DNA which play vital role in DNA repairing. In this, there are
various type of disease which is based on genetic level include mutation disorder, mutational
disorder and many more.
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REFERENCES
Books and Journals
Beltrán‐Corbellini and et. al., 2020. Acute‐onset smell and taste disorders in the context of
COVID‐19: a pilot multicentre polymerase chain reaction based case–control
study. European journal of neurology, 27(9), pp.1738-1741.
Kucirka and et. al., 2020. Variation in false-negative rate of reverse transcriptase polymerase
chain reaction–based SARS-CoV-2 tests by time since exposure. Annals of internal
medicine, 173(4), pp.262-267.
Li and et. al., 2020. Agarose native gel electrophoresis for characterization of
antibodies. International journal of biological macromolecules, 151, pp.885-890.
Liu and et. al., 2019. A comparison of plasmid DNA and mRNA as vaccine
technologies. Vaccines, 7(2), p.37.
Mettrick and et. al., 2020. Neutral–Neutral 2-Dimensional Agarose Gel Electrophoresis for
Visualization of E. coli DNA Replication Structures. In DNA Electrophoresis (pp. 61-
72). Humana, New York, NY.
Pandey and et. al., 2021. Recombinant DNA Technology for Sustainable Plant Growth and
Production. Harsh Environment and Plant Resilience: Molecular and Functional Aspects,
p.99.
Sefidi-Heris and et. al., 2020. Recent progress in the design of DNA vaccines against
tuberculosis. Drug Discovery Today, 25(11), pp.1971-1987.
Zare and et. al., 2017. Production of Chymosin using recombinant DNA technology.
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