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Assignment
SMK 5404
Laboratory Technique in
Medical Microbiology
NAME
I.C NO
: 890111 07 5185
MATRIC NO
: GS40862
TITLE
: FASTCLONING
EVALUATORS
CONCEPT of
MOLECULAR CLONING
Molecular Cloning: the basics
In molecular biology, DNA cloning is regarded as the most extensively used techniques
apart from polymerase chain reaction (PCR), blotting, sequencing, microarray and many
more. Basically, molecular cloning involves isolation of a DNA fragment from an organisms
complex chromosome and proceed with inserting into a molecular vector that is capable of
replicating the recombinant DNA into millions by multiplications of the competent cell.
Cloning of DNA may be outlined into five steps:
a) Cutting
of
DNA:
sequence-specific
scissors,
resulting
in
together
whereby
the
TYPES of
MOLECULAR CLONING
Molecular Cloning: the alternative methods
Although the original molecular cloning (described previously) is still widely used, due to
involvement of multiple steps, time consumption and difficulty for troubleshooting, the urgent
need for a simple and rapid techniques were identified. Over the past two decades, many
other alternative cloning techniques have been largely refined in order to deal with the
challenges faced in traditional method of cloning. Those methods include:
a) TA cloning:
Using the Thermus aquaticus (Taq) DNA polymerase which add 3-A overhang to the
ends of PCR product. A linearized T-vector with complementary 3-T overhang in
both ends is used to clone the amplified PCR product. This technique is commonly
employed when suitable recognition sites are not available, thereby surpassing the
activity of restriction enzymes (Zhou & Gomez-sanchez, 2000).
The cloning system require creation of an entry vector plasmid that acts as a donor of
the open reading frame (ORF). The ORF will be transferred from the entry vector into
a destination vector plasmid that provides components essential for expression. The
reaction is mediated by a robust enzyme system to excise the gene of interest and
integrate it into the destination vector, which then becomes expression clone
(Evdokimov, 2000).
DNA polymerase create a sticky ends of both the vector and insert without particular
sequence arrangements. The method is effective to run wide range of insert DNA
concentration and large PCR products such as 3 to 11kb. Uses enzymes with proof
reading activity, linking the DNA duplexes and consequently joining the vectors and
inserts precisely in sequence-independent (Berrow, Alderton, & Owens, 2009).
The method employed purification of the first round PCR products followed by further
round overlap PCR, which creates multiple bands, for producing linked cloning vector
and insert fragment. A simple and efficient way to clone an insert of choice into a
plasmid of choice without restriction endonucleases or DNA ligase (Bryksin &
Matsumura, 2011).
Despite, each techniques explained above each has its own limitations such as:
a) Purification of PCR products: time-consuming and require additional budget to
purchase purification kits.
b) PCR cycles: generally employed cycles are 25 to 30 cycles. Increased number of
PCR cycles, probably allow occurrence of random mutations, and thereby decreases
the cloning efficiency.
c) Loss of PCR product: possible dNTPs reduction in higher amount due to PCR
amplification or dNTPs dilution in process of purification capable of weakening the 3
ends protection against the activity of the DNA polymerase promoting the destruction
of PCR products.
METHODOLOGY ARTICLE on
FASTCLONING
FastCloning: the procedure
The insert and vector are subjected for 18 PCR amplification using a high fidelity
DNA polymerase. The primer pair of insert amplification has 16-base tails
overlapping the PCR-amplified vector ends. 5 l of PCR product were examined with
1% agarose gel electrophoresis running at 100 V for 30 min and stained with
ethidium bromide. The bands were visualized under a UV transilluminator. The
remaining of 45 l of unpurified PCR products of both cloning vector and insert were
mixed with ratio of 1:1.
c) Digestion of templates
1 l of DpnI restriction endonucleases was added into the mixture and subjected for
enzymatic digestion for 1 hour at 37C. In order to be compatible to enzyme
digestion, the parent DNA templates need to undergo methylation. It was suggested
that activity of the 3 exonuclease of high fidelity polymerase directly creates stick
ends for the overlapped regions of vector and insert during digestion. The restriction
enzyme destroys the methylated DNA templates.
Overlapped regions form a circular construct of nicks and proceed with repairing after
transformation into the competent host cell. 2 l of the digested vector-insert mixture
were added into 40 l of XL-10 Gold Escherichia coli cells. The mixture was
incubated on ice for 30 minutes. In order to induce cellular uptake, heat shock for 45
seconds at 42C was employed thereby forming pore onto the cells. 350 l of SOC
medium was then added to the mixture followed by 60 minutes of shaking at 37C at
350 rpm with a thermomixer.
KALIDASAN A/L VASODAVAN (GS40862)
The prepared content was placed onto the Luria Bertani (LB) agar plate containing
100 g/ml ampicillin. All plates were then incubated for 24 hours at 37C. Colonies
from each constructs were picked on next day for PCR confirmation and followed by
overnight culture of each clone for DNA mini-prep. All the cloned were finally
confirmed by automated DNA sequencing.
Attachment
Presentation slides