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UNIVERSITI PUTRA MALAYSIA

FACULTY OF MEDICINE & HEALTH SCIENCES

Master of Science (M.Sc.)


(Medical Microbiology)

Assignment
SMK 5404

Laboratory Technique in
Medical Microbiology
NAME

: KALIDASAN A/L VASODAVAN

I.C NO

: 890111 07 5185

MATRIC NO

: GS40862

TITLE

: FASTCLONING

EVALUATORS

: DR. SURESH KUMAR


DR. CHEE HUI YEE

SMK5404 - LABORATORY TECHNIQUE IN MEDICAL MICROBIOLOGY

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

restriction endonucleases, act as the


molecular

scissors,

resulting

in

generation of stick end.


b) Selecting a small DNA carrier: called
as cloning vector, typically of plasmids or
viral DNA that capable of self-replication
and carries antibiotic resistance genes.
c) Joining of DNA piece: DNA ligase
mediate the reaction between the cloning
vector and DNA fragment, covalently
joined

together

whereby

the

phosphodiester bond between phosphate


group of deoxyribose and hydroxyl group
of another deoxyribose, resulting in a
formation of recombinant DNA.
d) Recombinant DNA to a host cell by
transformation: selection of suitable
competent cells (usually bacterial cells
such as E. coli) whereby the cell supply
necessary enzymes for replication.
e) Selecting or identifying host cells: host
cells are plated on selective medium
containing antibiotic, screened for clones
of recombinant DNA.

KALIDASAN A/L VASODAVAN (GS40862)

Figure 1: Schematic illustration of


DNA cloning

SMK5404 - LABORATORY TECHNIQUE IN MEDICAL MICROBIOLOGY

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).

b) Ligation independent cloning using T4 DNA polymerase:

Involving the activity of 3-exonuclease T4 DNA polymerase to create 5-overhangs in


the ends of insert as well as complementary 5-overhangs in the ends of vector.
Requires particular sequence to create 15-base overhangs and a specific vector
containing a long stretch of sequence that do not have a particular deoxynucleotide
triphosphate (dNTP) (Jeong et al., 2012).

KALIDASAN A/L VASODAVAN (GS40862)

SMK5404 - LABORATORY TECHNIQUE IN MEDICAL MICROBIOLOGY

c) Gateway recombinant cloning:

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).

d) Latest ligation-independent cloning using kits (CloneEZ, In-Fusion):

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).

KALIDASAN A/L VASODAVAN (GS40862)

SMK5404 - LABORATORY TECHNIQUE IN MEDICAL MICROBIOLOGY

e) Overlap extension PCR cloning:

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.

KALIDASAN A/L VASODAVAN (GS40862)

SMK5404 - LABORATORY TECHNIQUE IN MEDICAL MICROBIOLOGY

METHODOLOGY ARTICLE on
FASTCLONING
FastCloning: the procedure

To circumvent the problems/limitations, a latest cloning method termed FastCloning was


developed. Fastloning is an easy, robust and efficient cloning technique based on PCR
amplification. The procedure of FastCloning is described as follows (Li et al., 2011):

a) PCR primers, reaction components and cycling parameters:


The primer pairs was designed to have temperature of annealing of 60C with Oligo
Analyzer 1.5. A 50 l total volume PCR reaction mix was prepared to include
components such as Phusion DNA polymerase, 0.5 l; Pfu Turbo or PfuUltra DNA
polymerase, 0.8 l; 10 buffer, 5 l; 2.5 mM dNTPs, 5 l; 10 ng of plasmid DNA
template and 5 pmol of both primers. The PCR parameters include initial
denaturation at 98C for 3 minutes, 18 cycles for denaturation at 98C for 10
seconds, annealing at 55C for 30 seconds, extension at 72C for 20 seconds, final
extension at 72C for 5 minutes and holding at 4C for infinite.

b) PCR amplification of vector and insert:

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.

KALIDASAN A/L VASODAVAN (GS40862)

SMK5404 - LABORATORY TECHNIQUE IN MEDICAL MICROBIOLOGY

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.

d) Transformation into competent cells:

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)

SMK5404 - LABORATORY TECHNIQUE IN MEDICAL MICROBIOLOGY

e) Plating onto suitable medium:

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.

FastCloning: the discussions


The method illustrated is reliable and effective thereby confer insertion of any DNA fragment
into a plasmid vector or into a cDNA of a vector at any desired site. Using only 18 PCR
cycles, and a high fidelity DNA polymerase, this method allow the vector and insert to be
amplified separately. The amplified insert end possess approximately 16-bases that overlaps
with the end of the amplified cloning vector. Following digestion of the mixture, it is directly
transformed into competent E. coli cells to obtain the desired clones. The advantages of
FastCloning surpass the techniques in comparison of other cloning methods. First, it does
not need a gel or purification kit for PCR product or vector. Second, there is no need of any
cloning kit or specialized enzyme for cloning. Furthermore, with decreased number of PCR
cycles, the chance of random mutations is largely reduced. In addition, this method is highly
efficient and reproducible. Finally, since this cloning method is also sequence independent, it
can be applied for construction of chimera, insertion or multiple mutations spanning a stretch
of DNA up to 120 bp.

KALIDASAN A/L VASODAVAN (GS40862)

SMK5404 - LABORATORY TECHNIQUE IN MEDICAL MICROBIOLOGY

Questions & Answers


1. How insert and vector are combined with ligation independent (without DNA
ligase) in FastCloning method?
Two assumptions can be made:
a) The primers for insert amplification have insert-specific sequences and additional
15 to 17 bases overlapping with the vector ends. Tailed PCR primer sets are
used to create complementary staggered overhangs on both DNA fragment and
cloning vector. Thus, it is hypothesized that the additional overlapping bases
allow directional cloning in a ligase-free manner.
b) The activity of the high fidelity DNA polymerase directly creates a sticky end for
the overlapped regions of vector and insert during the enzymatic digestion,
allowing formation of a circular construct with nicks. However, the mechanism is
not well understood.

2. What is high fidelity DNA polymerase and Phusion DNA polymerase?


The high-fidelity DNA polymerases, promote the 3' to 5' exonuclease activity, provide
more accurate amplification. Phusion DNA Polymerase generates PCR products with
speed and accuracy that are not unattainable with a single enzyme previosuly.
Additionally, Phusion DNA Polymerase is capable of amplifying long templates. Phusion
DNA polymerase can be used for the amplification and fusion reactions, so that
monitoring and optimization can be effective. The unique structure and characteristics of
Phusion DNA Polymerase make it a superior choice for cloning.

Attachment
Presentation slides

KALIDASAN A/L VASODAVAN (GS40862)