FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]
“A POLYNUCLEOTIDE, POLYPEPTIDE SEQUENCE AND METHODS
THEREOF”
Name and Address of the Applicant: CHROMOUS BIOTECH PVT. LTD, # 842, II
Floor, Sahakar Bhavan, A Block, Sahakar Nagar, Bangalore – 560092, INDIA.
Nationality: Indian
The following specification particularly describes the invention and the manner in which
it is to be performed.
2
TECHNICAL FIELD
The disclosure describes a novel nucleotide sequence encoding Taq polymerase enzyme
and the corresponding polypeptide sequence. The present disclosure involves genetic
engineering of the Taq polymerase enzyme to obtain a polymerase with an increased
level of processivity. The instant modification in the Taq polymerase causes a decrease in
the overall PCR reaction time and polymerizes 500 bp within a second. This genetically
engineered Taq polymerase has its applications in Biotechnological industry and is
suitable to be used in the different types of PCRs where conventionally, the wild type Taq
polymerase enzyme is employed.
BACKGROUND OF THE DISCLOSURE
Taq polymerase is a thermostable DNA polymerase originally isolated from the
thermophilic bacterium Thermus aquaticus in 1965. T. aquaticus is a bacterium that lives
in hot springs and hydrothermal vents. Taq polymerase was identified as an enzyme able
to withstand the protein-denaturing conditions (high temperature) required during PCR.
This enzyme’s optimum temperature for activity is 75-80°C, with a half-life of 9 minutes
at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at
72°C.
Taq DNA polymerase catalyzes the incorporation of dNTP’s into DNA. It requires a
DNA template, a primer terminus, and the divalent cation Mg++. Taq polymerase
contains a polymerization dependent 5'-3' exonuclease activity. It does not have a 3'-5'
exonuclease activity and thus no proof-reading function. Due to its considerable
importance in PCR and other related applications, Taq polymerase has evolved to be one
of the most researched and highly useful enzymes of the present times.
Taq polymerase is composed of 832 amino acids and has three separate functional
domains: the nuclease domain, the nucleotide editing domain and the polymerase
domain.
3
The nuclease domain is composed of amino acid residues 1-290 and is responsible for
deletion of the template DNA / primer-dimers, etc. The nuclease activity is known to be
weaker with comparison to other known nucleases and is mostly neutralized by a strong
polymerase activity present in the other part of the protein.
The nucleotide editing domain is composed of residues 291-423 and is responsible for the
enzyme fidelity. Incorrect incorporations of bases by the polymerase are detected and
repaired by the domain. The Taq Polymerase is known to have fidelity of 66 x 10-6 (66
mutations introduced per million bases incorporated) in comparison to Pfu polymerase
which has fidelity of 2.8 x 10-6 (2.8 mutations introduced per million bases incorporated).
The polymerase domain is responsible for incorporating new nucleotides into the DNA
template and is composed of residues 424-832. The enzyme is known to have an
extension rate of 35-100 nt/sec at 75°C.
Apart from the above mentioned functions related to the Taq Polymerase, the enzyme
also has an important function termed processivity. Processivity refers to the number of
nucleotides incorporated by a polymerase before it dissociates. The wild-type enzyme
requires approximately about 1 minute per 1 Kb DNA fragment polymerization. Thus, a
standard PCR cycle condition for amplification of a 1Kb DNA fragment lasts between 2
hours and 30 minutes to about 3 hours based on the specific PCR machine used. Hence,
to reduce the total duration of the PCR process, Fast-PCRs are suggested where the PCR
Machine can change temperature (ramp time), making it a very fast process and thus
reducing the PCR run time.
The Taq DNA polymerase is a thermo-stable enzyme that can amplify any target DNA
about billion-times even after 30 cycles of PCR. Due to all these reasons the Taq
polymerase enzyme has become indispensable in the Biotechnological industry.
4
Some of the limitations of the existing technology:
• Longer PCRs, where about 7-10 Kb DNA fragment are amplified, the PCR process
requires about 5-7 hours for completion.
• The longer time required to complete the entire PCR process, limits the usage of the PCR
machine.
• The longer the PCR process, the higher is the chance of failure of the reaction because of
loss of activity of the Taq polymerase enzyme.
• Failures are known much later for repeat reactions / modified protocols to follow.
• Molecular diagnostic applications require much longer and only “next day” report is
feasible. Immunological tests are preferred for getting results the same day.
SUMMARY OF THE DISCLOSURE
An illustrative embodiment of the present disclosure relates to a nucleotide sequence
encoding DNA polymerase as set forth in Sequence Id No. 1.
An illustrative embodiment of the present disclosure relates to a polypeptide sequence as
set forth in Sequence Id No. 2.
An illustrative embodiment of the present disclosure relates to a genetically modified
DNA polymerase comprising polypeptide sequence as set forth in Sequence Id No. 2
alongwith storage buffer.
According to another aspect of the present disclosure, the embodiment relates to a
recombinant vector comprising nucleotide sequence set forth in Sequence Id No.1.
According to another aspect of the present disclosure, the embodiment relates to a
recombinant host cell, transformed by introduction of a vector comprising nucleotide
sequence set forth in Sequence Id No.1.
An illustrative embodiment of the present disclosure relates to primers as set forth in
Sequence Id No. 3 and Sequence Id No 4.
According to another illustrative embodiment, the present disclosure relates to a method
of amplification of nucleotide sequences, said method comprising step of amplifying the
nucleotide sequence by a DNA polymerase as set forth in Sequence Id No.1.
According to another illustrative embodiment, the present disclosure relates to a PCR
reaction mixture, said mixture comprising sample to be detected, probes, primers, DNA
5
polymerase comprising the nucleotide sequence set forth in Sequence Id No. 1 and
nucleic acid amplification reagents.
According to another illustrative embodiment, the present disclosure relates to a kit
comprising probes, primers, DNA polymerase having nucleotide sequence set forth in
Sequence Id No.1 or polypeptide sequence set forth in Sequence Id No.2 and
amplification reagents.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect,
reference will now be made to exemplary embodiments as illustrated with reference to
the accompanying figures. The figure together with a detailed description below, are
incorporated in and form part of the specification, and serve to further illustrate the
embodiments and explain various principles and advantages, in accordance with the
present disclosure where:
Figure 1 shows mutation introduction protocol.
Figure 2 shows amplification plot of one of the mutants.
Figure 3 shows gel Electrophoresis of 5 Kb fragment amplified from λ-DNA.
Lane M – Chromous SuperMix Ladder.
Lane 1 – Wild-type Enzyme, Standard PCR.
Lane 2 - Mutant Enzyme, Standard PCR.
Lane 3 - Wild-type Enzyme, Fast PCR.
Lane 4 - Mutant Enzyme, Fast PCR.
Figure 4 shows mutated Taq polymerase gene cloning.
Figure 5 shows gel Electrophoresis of 5 Kb fragment amplified from λ-DNA.
Lane M – SuperMix DNA Ladder.
Lane 1 – Wild-type Enzyme, Standard PCR.
Lane 2 - Mutant Enzyme, Standard PCR.
Lane 3 - Wild-type Enzyme, Fast PCR.
Lane 4 - Mutant Enzyme, Fast PCR.
Figure 6 shows real-Time PCR Data.
6
Figure 7 shows gel Electrophoresis of 1.5 Kb fragment amplified from Bacterial gDNA.
Lane M – Chromous SuperMix Ladder.
Lane 1 – Wild-type Enzyme, Standard PCR.
Lane 2 - Mutant Enzyme, Standard PCR.
Lane 3 - Wild-type Enzyme, Fast PCR.
Lane 4 - Mutant Enzyme, Fast PCR.
Figure 8 shows real-Time PCR Data.
Figure 9 shows gel Electrophoresis of 5 Kb fragment amplified from λ-DNA.
Lane M – Chromous SuperMix Ladder.
Lane 1 - Wild-type Enzyme, Standard PCR.
Lane 2 - Mutant Enzyme, Standard PCR.
Lane 3 - Wild-type Enzyme, Fast PCR.
Lane 4 - Mutant Enzyme, Fast PCR.
DETAILED DESCRIPTION
The present disclosure relates to a nucleotide sequence encoding DNA polymerase as set
forth in Sequence Id No. 1.
In an embodiment of the present disclosure the DNA polymerase is mutated Taq DNA
polymerase.
In another embodiment of the present disclosure the mutation is a nucleotide substitution
at 1337th position.
The present disclosure relates to a polypeptide sequence as set forth in Sequence Id No.
2.
In an embodiment of the present disclosure the polypeptide sequence is a mutated Taq
DNA polymerase polypeptide.
In another embodiment of the present disclosure the polypeptide sequence comprises a
mutation at 446th amino acid residue.
In yet another embodiment of the present disclosure the polypeptide sequence
corresponds to nucleotide sequence set forth in Sequence Id No. 1.
The present disclosure relates to a genetically modified DNA polymerase comprising
polypeptide sequence as set forth in Sequence Id No. 2 along with storage buffer.
7
In an embodiment of the present disclosure the storage buffer is Taq storage buffer
comprising Tris, KCl, EDTA, Tween20,NP-40, DTT and Glycerol.
In another embodiment of the present disclosure the Taq storage buffer preferably
comprises about 50mM Tris at pH 8.0, about 50mM KCl, about 1mM EDTA, about 0.5%
Tween20, about 0.5% NP-40, about 1mM DTT and about 50% Glycerol.
In an embodiment of the present disclosure the DNA polymerase is a Taq DNA
polymerase.
In another embodiment of the present disclosure the polypeptide sequence corresponds to
nucleotide sequence set forth in Sequence Id No. 1.
In yet another embodiment of the present disclosure the polypeptide sequence comprises
a mutation at the 446th amino acid residue and the nucleotide sequence has a nucleotide
substitution at 1337th position.
In still another embodiment of the present disclosure the DNA polymerase has
processivity of amplifying about 500 base pairs per second.
In still another embodiment of the present disclosure the DNA polymerase can be used in
combination with other modifying enzymes selected from a group comprising Reverse
Transcriptases selected from a group consisting AMV-RT and MMuLV-RT and Proof
Reading Polymerase preferably Pfu Polymerase.
The present disclosure relates to a recombinant vector comprising nucleotide sequence
set forth in Sequence Id No.1.
In an embodiment of the present disclosure the vector is pUC 18.
In another embodiment of the present disclosure the nucleotide sequence encodes a DNA
polymerase having a nucleotide substitution at 1337th position.
The present disclosure relates to a recombinant host cell, transformed by introduction of a
vector comprising nucleotide sequence set forth in Sequence Id No.1.
In an embodiment of the present disclosure the host cell is selected from a group
comprising E. coli JM series, E. coli DH series and E. coli with lac operon system, and
wherein the vector is pUC 18.
In another embodiment of the present disclosure the nucleotide sequence encodes a DNA
polymerase having a nucleotide substitution at 1337th position.
8
The present disclosure relates to primers as set forth in Sequence Id No. 3 and Sequence
Id No. 4.
In an embodiment of the present disclosure the primer set forth in Sequence Id No 3 is
forward primer and the primer set forth in Sequence Id No. 4 is reverse primer.
In another embodiment of the present disclosure the primer is used for introducing
mutations in DNA polymerase by site directed random mutagenesis.
The present disclosure relates to a method of amplification of nucleotide sequences, said
method comprising step of amplifying the nucleotide sequence by a DNA polymerase as
set forth in Sequence Id No.1.
In an embodiment of the present disclosure the DNA polymerase is a mutated Taq DNA
polymerase having a nucleotide substitution at 1337th position.
In another embodiment of the present disclosure the DNA polymerase has processivity of
amplifying about 500 base pairs per second.
The present disclosure relates to a PCR reaction mixture, said mixture comprising sample
to be detected, probes, primers, DNA polymerase comprising the nucleotide sequence set
forth in Sequence Id No. 1 and nucleic acid amplification reagents.
In an embodiment of the present disclosure the sample comprises genetic material
selected from a group comprising Plants, Animals, Bacteria, Fungi, Algae and Virus.
In another embodiment of the present disclosure the DNA polymerase is a mutated Taq
DNA polymerase having a nucleotide substitution at 1337th position.
In yet another embodiment of the present disclosure the DNA polymerase has
processivity of amplifying about 500 base pairs per second.
The present disclosure relates to a kit comprising probes, primers, DNA polymerase
having nucleotide sequence set forth in Sequence Id No.1 or polypeptide sequence set
forth in Sequence Id No.2 and amplification reagents.
In an embodiment of the present disclosure the DNA polymerase is a mutated Taq DNA
polymerase having a nucleotide substitution at 1337th position.
In another embodiment of the present disclosure the DNA polymerase has enhanced
processivity of amplifying about 500 base pairs per second.
In yet another embodiment of the present disclosure the kit is used for performing PCR
amplifications.
9
In an embodiment of the present disclosure the PCR is selected from a group comprising
Standard PCR, Real time PCR, Reverse Transcription PCR and Long PCR
Taq polymerase is composed of 832 amino acids broken down into three separate
functional domains. The nuclease domain is composed of amino acid residues 1-290, the
nucleotide editing domain is composed of residues 291-423 and the polymerase domain
has residues 424 to 832. The junction region (the region between the editing domain and
the polymerase domain) i.e., the region between 400 and 450 amino acids was chosen for
the study. Random mutants were developed and screened performing PCR where the
mutated enzymes were challenged for faster processivity (amplification of 5 kb within 30
sec extension time). One particular mutant showed higher amplification rate when
compared to the wild type enzyme and sequencing the gene confirmed the presence of
mutation at 446th amino acid (A to V). This mutation was subsequently reverted and the
polymerase activity was found to be similar to the wild-type Taq polymerase.
Taq polymerase mutants are developed by the site directed mutagenesis protocol,
following conventional steps. The thermo-stable PCR enzyme, Taq DNA Polymerase, is
genetically engineered by site-directed random mutagenesis protocol to randomly alter
specific domains of the enzyme.
Several sets of mutants are developed and screened for enhanced processing activities.
While there are loss of activities in some, the present Mutant enzyme showed dramatic
improvement in processivity.
Standard PCRs, amplification of GC-rich regions, amplification of Long-fragments (upto
25 kb) and multiplex PCR reactions are tried for this Mutant enzyme. All results showed
identical performance for the enzyme with a dramatic reduction of polymerization time.
The present Mutant enzyme could amplify 500 bp fragments within 1 sec polymerization
time and 1 kb fragment within 2 seconds. Other steps: the denaturation and annealing
times could also be reduced by 50-70%. As a result, a standard 500 bp amplification
10
could be completed within 35 minutes in a normal PCR machine (ABI-2720) and
amplification is observed in a Real-Time Fast-PCR machine (StepOne RT-PCR, ABI)
within 9 minutes. Similarly, 1 kb amplification could be completed within 48 minutes
and 20 minutes respectively.
• PROTOCOL FOLLOWED FOR SITE-DIRECTED RANDOM MUTAGENESIS
Standard Site-directed mutagenesis protocols are available that introduce changes at a
particular position on the gene. Most protocols are PCR-based and the mutants are
screened by restriction digestion (site introduced along with the desired mutation for
screening) or by sequencing.
Standard Random mutagenesis protocols are available where a gene is randomly
mutated, again using mostly PCR-based protocols. The mutants are developed and
screened for a particular trait of interest. Desired mutants are sequenced to find out
particular mutation(s) responsible for a modified trait.
For site-directed random mutagenesis, a domain of interest/ region on the gene is
randomly mutated to effectively study the significance of the domain/ region. Also, the
modified domains might display altered performances, better than or inferior to the wild
type molecule.
For the current studies, the domain responsible for processivity of the wild type Taq
polymerase between amino acids 400 and 450 is targeted for site-directed random
mutagenesis. Primer set are designed and synthesized to specifically amplify the said
domain under “erroneous” PCR conditions that introduces mutations in the region. The
template used for primer designing is the Taq polymerase gene cloned into an Expression
vector under Lac promoter-based expression cassette. Particularly, Primer sequences as
set forth in Sequence ID No. 3 and 4 are used. The Sequence ID No. 3 corresponds to
Forward primer and the Sequence ID No. 4 corresponds to Reverse Primer. PCR is
performed and the PCR amplicons, a combination of amplicons with differential mutated
sequences, are then cloned back into the gene missing the domain. Around 200
11
independent clones were developed and confirmed by usual gene cloning techniques
(plasmid retardation and restriction digestion/ release of insert of expected size). Figure 1,
illustrates the site-directed random mutation introduction protocol. The Primer sequences
designed and as set forth in Sequence ID No. 3 and 4 are used for performing first PCR,
using buffer with about 200μM dGTP and dATP, about 1mM dTTP and dCTP, about
7mM MgCl2 and about 300 μM MnCl2. The First PCR Product with random mutations is
obtained after the First PCR process. Thereafter, the second PCR process is conducted
using the First PCR products as the primers for the second PCR process. The Second
PCR process results in mutants incorporated with random mutations. These random
mutants are thereafter screened and sequencing is done on screened mutants.
• PROTOCOL FOLLOWED FOR SCREENING THE MUTANT: BASED ON PCR
AND REAL-TIME PCR
All the clones obtained are screened for such clones that can amplify a 5 kb fragment
under FAST-PCR cycle conditions. The PCR-based screening was performed on a Real-
Time PCR platform where a lower Ct-value would distinguish the mutant(s) with faster
processivity in comparison with the wild type clones. The FAST-PCR is such a PCR
wherein the PCR cycle conditions are slightly modified to be in accordance with the
instant mutant Taq Polymerase and the protocol followed is illustrated in the table 1
below:
Table 1
94 ºC 94 ºC 55 ºC 72 ºC 72 ºC
2 min 5 sec 5 sec 30 sec 1 min
1 cycle 40 cycles 1 cycle
Significantly, the extension cycle that requires duration of about 5 minutes during
standard PCR amplification of a 5 Kb sequence, is kept at 30 seconds (highlighted). This
was done in order to screen the mutants which showed high processivity. The
amplification plot of one of the mutants with lower Ct-value is shown in the figure 2.
12
The same mutant is also screened for performance on a standard PCR machine (model
ABI-2700), the standard PCR cycle conditions followed is depicted in Table 2.
Thereafter, Gel electrophoresis is done, the gel photograph is depicted in figure 3.
Table 2
94 ºC 94 ºC 55 ºC 72 ºC 72 ºC
2 min 30 sec 30 sec 5 min 5 min
1 cycle 30 cycles 1 cycle
The results depicted in figure 3, show good amplification by the instant mutant Taq
polymerase of 5 kb amplicon, under Fast PCR condition (with 30 seconds of extension
time) while other clones as well as the wild-type clone fails to amplify. The results also
demonstrate that the instant mutant as well as the wild-type clone, both could amplify the
5 kb fragment under standard PCR cycle conditions as well.
The mutant clone with faster processivity is subsequently characterized by sequencing
the polymerase gene using vector-specific primers and gene-specific primers to detect
the mutation(s) present. The sequence data is aligned and analyzed by BLAST (NCBI).
The mutation detected is highlighted in the nucleotide as well as the polypeptide
sequence as set forth in Sequence ID Nos. 1 and 2.
• VECTOR AND HOST CELL
The instant mutant Taq polymerase enzyme nucleotide sequence as set forth in Sequence
ID No. 1, is introduced into a vector, preferably pUC 18 (as depicted in figure 4).
Thereafter, a host cell is transformed by introduction of the recombinant vector
comprising the nucleotide sequence set forth in Sequence ID No.1. E. coli strains with lac
operon system present in them are selected as host cells for transformation by the
recombinant vector. The E. coli hosts used for transformation in the present study are
selected from E. coli JM and E. coli DH series, preferably E. coli DH5α strain cells are
used. Hence, transformed E. coli DH5α strain cells are obtained, these cells are induced
13
and the protein purified. The activity of the purified protein is analysed by Activity Assay
and the active enzyme is isolated from the transformed cells using the following protocol.
a) Inoculate 1 colony of the transformed E. coli cells into a 5 ml LB containing 100 ppm
Ampicillin. The culture is grown for 16 hours at 37ºC on a shaker incubator at 160 rpm.
b) Thereafter, 1% of the 16 hour old/grown culture is inoculated into a 500ml LB containing
100 ppm Ampicillin. The culture is grown at 37ºC on a shaker incubator at 160 rpm, until
the OD attains a value of 0.5 at 600nm.
c) Once the OD has attained a value of 0.5, the culture is induced by addition of 5mM
IPTG.
d) After addition of IPTG, the culture is grown for a period of 12 hours at 37ºC on a shaker
incubator at 160 rpm.
e) The induced cells were then spun down at 6,000 rpm for 10 min and lysed by sonication.
f) The sonicated sample was spun down at 10,000 rpm for 30 min in a 4ºC refrigerated
centrifuge.
g) The supernatant is taken and placed in a water bath at 80ºC for 30 minutes.
h) The incubated sample is spun down at 10,000 rpm for 30 minutes at Room temperature.
i) Then, the supernatant is taken and to the supernatant 30% ammonium sulfate is added
while stirring on a magnetic stirrer at 4ºC. The sample was stirred for 30 minutes at 4ºC.
j) The sample is spun down at 10,000 rpm for 30 minutes in 4ºC refrigerated centrifuge.
k) The supernatant is discarded and the pellet is suspended in 5 ml of Taq Pre-dialysis
buffer (50 mM Tris pH 8.0, 50 mM KCL, 1 mM EDTA, 0.5 % Tween20, 0.5% NP-40
and 1 mM DTT).
l) The Suspended enzyme is finally dialyzed against Taq storage buffer and stored in the
same. The Storage buffer is preferably Tris-EDTA Buffer comprising 50 mM Tris pH
8.0, 50 mM KCL, 1 mM EDTA, 0.5 % Tween20, 0.5% NP-40, 1 mM DTT and 50 %
Glycerol. Further, as the scope of the present disclosure is non-limiting in nature, the
instant purified Taq polymerase enzyme can be dialyzed/stored in various storage buffers
such as but not limiting to those provided above. Further, all the possible storage buffers
useful in similar biotechnological applications and conventionally known fall under the
purview of the present disclosure.
14
The biological activities of individual enzyme clones (probable mutants) were checked/
screened by Real Time PCR assay. The Biological material present in the instant
disclosure in the form of host cell comprising genetically modified vector was deposited
at the International Depository – Microbial Type Culture Collection & Gene Bank,
Chandigarh. The deposited host cell was assigned MTCC Number MTCC 5546.
• DETAILS OF STANDARD AND FAST PCR CYCLE CONDITIONS
The Standard PCR protocols comprise of 3 steps: Initial denaturation, Cyclic change of
temperatures and a final Polymerization step.
The initial denaturation step in standard PCR protocol is at 94ºC for 2 minutes which
remains unchanged for the Fast PCR format as well. However, the “cyclic change of
temperature” steps vary widely between the two protocols. In standard PCR protocols,
the typical steps for a 5 kb amplification reaction are captured in the table 3 below:
Table 3
94 ºC 55 ºC 72 ºC
30 seconds 30 seconds 5 minutes
40 cycles
Thus, each cycle would last for 6 minutes (plus the ramp time) and around 5 hrs for 40
PCR cycles. For Fast PCR protocols, using the mutant Taq polymerase, the typical steps
for a 5 kb amplification reaction are provided in the table 4 below:
Table 4
94 ºC 55 ºC 72 ºC
5 seconds 5 seconds 30 seconds
40 cycles
Thus, each cycle would last for 40 seconds (plus the ramp time) and around 40 min for 40
cycles.
15
• SCIENTIFIC APPLICATIONS
1. Routine PCRs that require two and half hours to three hrs can be completed within 40
minutes.
2. The enzyme can amplify long-DNA fragments of 5-7 Kb within 2 hours.
3. Under Fast PCR conditions, the yield of amplicons for long-PCR is higher by ~50%
or more. However, the yields are comparable under standard PCR conditions.
4. Crude samples like bacterial Colony, blood stains, Soil and human hair follicles can
be used directly for Fast PCR.
5. Fast format in Real-Time PCR reactions would save time. The Ct-values would be
much lower than standard PCRs leading to early detections and reduced machinetime.
6. Colony PCR is performed for screening of clones (insert size 500 bp to 1 kb) on
normal PCR machine (ABI-2720). 96 Colony to PCR screening was completed in ~
90 mins.
7. Multiplex Fast-PCR (a protocol to amplify multiple fragments together in a single
reaction) has been tried using E. coli genomic DNA. Products of size 1 and 1.5 kb
were successfully amplified within 45 minutes.
• INDUSTRIAL APPLICATIONS
1. Molecular diagnostic applications for detection of virus / bacteria can be completed
within 10-15 minutes on a Real-time PCR.
2. Increased efficiency in machine-usage; upto 5 times more runs per day per machine
for both PCR and Real-Time PCR machines.
3. On-site disease Detection based on Fast PCR protocols. On Real-Time PCR format,
detection of target could be completed as early as 10 minutes, thus, making disease
detection possible on-site.
The present disclosure is further elaborated with the help of following examples and
associated figures. However, these examples should not be construed to limit the scope of
the present disclosure. Further, as the scope of the present disclosure is non-limiting in
nature, the instant modified Taq polymerase enzyme can be applied to various other
16
biotechnological applications such as but not limiting to those illustrated in the following
examples. Further, all the possible amplification experiments involved in all such
biotechnological applications fall under the purview of the present disclosure. The instant
Taq polymerase can also be used in combination with other modifying enzymes such as
but not limiting to Reverse Transcriptases and Proof-reading polymerases. All possible
modifying enzymes conventionally known in the art, fall under the purview of the present
disclosure.
• EXAMPLES
EXAMPLE 1: Routine PCRs that requires two and half hours to three hrs can be
completed within 40 minutes.
1.1 Amplification of 500 bp from plasmid
PCR Cycle conditions for Standard PCR are provided in the table 5 below:
Table 5:
940C 940C 550C 720C 720C 40C
2 min 30 sec 30 sec 30 sec 15 min ∞
35 cycles
PCR Cycle conditions for Fast PCR using the instant mutated Taq Polymerase, are
provided in the table 6 below:
Table 6
940C 940C 550C 720C 720C 40C
2 min 5 sec 5 sec 1 sec 1 min ∞
35 cycles
Identical sequence-specific primers were used for both standard as well as Fast PCR
protocols. Identical PCR amplicons were observed, the difference being: the standard
17
PCR protocol required 2 hrs and 30 min while the Fast PCR was completed within 30
min.
1.2 Amplification of 1 Kb from plasmid
PCR Cycle conditions for Standard PCR are provided in the table 7 below:
Table 7
940C 940C 550C 720C 720C 40C
2 min 30 sec 30 sec 1 min 15 min ∞
35 cycles
PCR Cycle conditions for Fast PCR using the instant mutant Taq Polymerase, are
provided in the table 8 below:
Table 8
940C 940C 550C 720C 720C 40C
2 min 5 sec 5 sec 2 sec 1 min ∞
35 cycles
Identical sequence-specific primers were used for both standard as well as Fast PCR
protocols. Identical PCR amplicons were observed, the only difference being that the
standard PCR protocol required longer duration of time while the Fast PCR was
completed in a shorter duration of time.
EXAMPLE 2: The enzyme can amplify long-DNA fragments of 5-7 kb within 2 hours.
Long PCR under standard PCR cycle conditions.
2.1 Amplification of 3 to 5 kb from Lambda DNA
Standard PCR Cycle Conditions are provided in the table 9 below:
18
Table 9
940C 940C 550C 720C 720C 40C
2 min 30 sec 30 sec 3-5 min 15 min ∞
35 cycles
Fast PCR Cycle Condition using the instant mutant Taq Polymerase, are provided in the
table 10 below:
Table 10
940C 940C 550C 720C 720C 40C
2 min 5 sec 5 sec 30 sec 30 sec ∞
35 cycles
Identical sequence-specific primers were used for both standard as well as Fast PCR
protocols, for the amplification of long DNA. Further the resultant PCR amplicons
observed were also identical; the only difference was that the standard PCR protocol
required longer duration of time while the Fast PCR was completed in a short duration of
time.
2.2 Amplification of 7 kb from Lambda DNA
Standard PCR Cycle Conditions followed are provided in the table 11 below:
Table11
940C 940C 550C 720C 720C 40C
2 min 30 sec 30 sec 7 min 15 min ∞
35 cycles
Fast PCR Cycle Conditions followed are depicted in the table 12 below:
Table 12
940C 940C 550C 720C 720C 40C
2 min 5 sec 5 sec 2 min 2 min ∞
35 cycles
19
Identical sequence-specific primers were used for both standard as well as Fast PCR
protocols, for the amplification of long DNA fragments. Further, the resultant PCR
amplicons observed were also identical, the only difference was that the standard PCR
protocol required longer duration of time while the Fast PCR was completed in a short
duration of time, for the amplification of same length Long DNA fragments.
EXAMPLE 3: Under Fast PCR conditions, the yield of amplicons for long-PCR is
higher by ~50% or more. However, the yields are comparable under standard PCR
conditions.
In the figure 5, the yields of 5 kb fragment amplicon amplified from Lambda-DNA,
under standard PCR conditions using the wild-type enzyme is depicted in lane 1. While
the lane 2 depicts amplification using the instant mutant Taq enzyme under similar
standard conditions. Both these lane 1 and lane 2 bands are comparable. However, the
wild-type enzyme under Fast PCR conditions does not show activity as the PCR
conditions are different and hence no band is observed in the lane 3, in other words the
amplicon is not visible under Fast PCR conditions. On the other hand, as can be observed
from the figure 5, the instant mutant Taq polymerase shows activity in the Fast PCR
conditions as well.
• Crude samples like bacterial Colony, blood stains, Soil and human hair follicles can
be used directly in Fast PCR.
For experiments where the template DNA source is scarce, column-based high-purity
protocols are difficult to follow. However, rapid genomic DNA isolation protocols from
scarce sources, if worked reproducibly on PCR, would make detection of genes/
genotypes possible.
EXAMPLE 4: Colony PCR: (for bacteria)
Isolation of genomic DNA/ plasmid DNA from a single bacterial colony and screening
by Fast PCR protocols: Inset size 500 bp to 1 kb.
20
Fast PCR Cycle Conditions followed are provided in the table 13 below:
Table 13
940C 940C 550C 720C 720C 40C
2 min 5 sec 5 sec 2 sec 1 min ∞
35 cycles
Sequence-specific primers were used for amplification of DNA obtained from Bacteria
using the Fast PCR protocols. PCR amplicons were observed within the short duration of
the Fast PCR conditions.
EXAMPLE 5: Fast PCR from human hair follicle and blood smears; Here, amplification
of 500 bp human CRRH gene was done.
Fast PCR Cycle Conditions followed are provided in the table 14 below:
Table 14
940C 940C 550C 720C 720C 40C
5 min 10 sec 10 sec 5 sec 2 min ∞
35 cycles
Sequence-specific primers were used for amplification of DNA obtained from human
hair follicle and blood smears using the Fast PCR protocols. PCR amplicons were
observed within the short duration of the Fast PCR conditions.
Fast PCR format in Real-Time PCR reactions would save time. The Ct-values would be
much lower than those observed with standard PCRs, leading to early detections and
reduced machine-time.
EXAMPLE 6: Amplification of 1.7 – 2 kb 18 S gene fragment from plant and
human genomic DNA:
21
Fast PCR Cycle Conditions followed are provided in the table 15 below:
Table 15
940C 940C 550C 720C 720C 40C
5 min 10 sec 10 sec 20 sec 2 min ∞
35 cycles
Sequence-specific primers were used for amplification of DNA obtained from Plant and
human sources, using the Fast PCR protocols. PCR amplicons were observed within the
short duration of the Fast PCR condition. The PCR amplicons could be sequenced
directly (without Clean-up reactions) and good quality sequence data was obtained.
EXAMPLE 7: PCR Amplification and Cloning of 16S rDNA: (Bacterial Identification
and Population analysis)
Fast PCR Cycle Conditions followed are provided in the table 16 below:
Table 16
940C 940C 550C 720C 720C 40C
2 min 5 sec 5 sec 10 sec 1 min ∞
35 cycles
Sequence-specific primers were used for amplification of DNA using the Fast PCR
protocols. The 16S rDNA region (~1.5 kb) was amplified from more then 100 bacteria.
The amplified products were cloned into T/A cloning vector and the sequences
confirmed. The efficiency of cloning reactions (ligation, transformation etc.) attained was
comparable to that of PCR amplicons obtained from standard PCR protocols.
EXAMPLE 8: Amplification of ITS region from Yeast and other fungal samples (Fungal
Identification)
Genomic DNA was isolated from yeast and other fungal samples and Fast PCR protocols
were subjected to the same. Sequence-specific primers were used for amplification of the
22
DNA obtained. PCR amplicons were observed within the short duration of the Fast PCR
conditions, specifically ~500 bp ITS region was amplified and the sequences confirmed.
The Fast PCR Cycle Conditions followed are given in the table 17 below:
Table 17
940C 940C 550C 720C 720C 40C
2 min 5 sec 5 sec 2 sec 2 min ∞
35 cycles
EXAMPLE 9: Multiplex PCR Amplification of 500 bp, 1 kb and 1.5 kb amplicons by
Fast PCR protocols
Primer sets were designed for 3 independent PCR amplifications and were multiplexed
for amplification of all 3 amplicons using Fast PCR cycle conditions. The results
obtained were comparable to those obtained when similar amplification is done in a
Standard Multiplex PCR.
Fast PCR Cycle Conditions used are provided in the table 18 below:
Table 18
940C 940C 550C 720C 720C 40C
2 min 5 sec 5 sec 10 sec 2 min ∞
35 cycles
EXAMPLE 10: SNP analysis of human CRRH gene
A ~500 bp DNA fragment was amplified from human sputum using Fast PCR protocols
and then sequenced to analyse presence of SNPs.
The Fast PCR Cycle Conditions followed are provided in the table 19 below:
Table 19
940C 940C 550C 720C 720C 40C
5 min 10 sec 10 sec 2 sec 2 min ∞
35 cycles
23
Sequence-specific primers were used for amplification of DNA obtained from human
sputum, using the Fast PCR protocols. PCR amplicons were observed within the short
duration of the Fast PCR condition. The amplification results observed were comparable
to the amplification observed under similar Standard PCR conditions.
EXAMPLE 11:
Real-Time PCR based data / plots to demonstrate faster processing of the Taq polymerase
of the present disclosure.
To elucidate the characteristic differences between the wild type Taq polymerase and the
instant mutant, standard and Fast PCR were performed. Amplification of DNA fragments
of 1.5 Kb and 5.0 Kb were done. The PCR cycle conditions for both the Standard and
Fast PCR are mentioned below.
11.1 PCR Cycle Conditions for amplification of 1.5 kb fragment from bacterial
genomic DNA:
Fast PCR Cycle Conditions using the Taq Polymerase are provided in the table 20 below:
Table 20
94 ºC 94 ºC 55 ºC 72 ºC 72 ºC
2 min 5 sec 5 sec 30 sec 1 min
1 cycle 40 cycles 1 cycle
Standard PCR Conditions followed are provided in the table 21 below:
Table 21
94 ºC 94 ºC 55 ºC 72 ºC 72 ºC
2 min 30 sec 30 sec 5 min 5 min
1 cycle 30 cycles 1 cycle
Both the wild-type Taq Polymerase enzyme and instant mutant Taq Polymerase enzyme
were subjected to Standard PCR as well as Fast PCR conditions. The results obtained in
each case are elaborated below.
24
Results:
1.5 kb Amplification:
There was no significant difference observed with respect to the Ct-values during the
amplifications using both wild type and mutant Taq Polymerase enzyme under standard
PCR cycle conditions. However, there were significant differences in Ct-values when
both the enzymes were subjected to Fast PCR cycle conditions.
The Ct-values in the Fast PCR cycle conditions for wild type enzyme was ~27 cycles
while the same for the mutant enzyme was ~14 cycles. The differences in Ct-values are
very significant in terms of processivity and indicate that the mutant enzyme performed
faster under Fast PCR cycle conditions generating ~ 8000 times more amplicons than the
wild type enzyme. The above aspects are depicted in figure 6, as Real-Time PCR Data.,
and the corresponding Gel Electrophoresis analysis is depicted in figure 7.
11.2: PCR Cycle Conditions for amplification of 5.0 kb fragment from bacterial
genomic DNA:
Fast PCR Conditions followed are depicted in the table 22 below:
Table 22
94 ºC 94 ºC 55 ºC 72 ºC 72 ºC
2 min 5 sec 5 sec 30 sec 1 min
1 cycle 40 cycles 1 cycle
Standard PCR Conditions followed are depicted in the table 23 below:
Table 23
94 ºC 94 ºC 55 ºC 72 ºC 72 ºC
2 min 30 sec 30 sec 5 min 5 min
1 cycle 30 cycles 1 cycle
Both the wild-type Taq Polymerase enzyme and instant mutant Taq Polymerase enzyme
were subjected to Standard PCR as well as Fast PCR conditions. The results obtained in
each case are elaborated below.
25
Results:
5 kb Amplification:
There were significant differences observed in the Ct-values for 5 kb DNA fragment
amplifications under standard PCR cycle conditions. Further, there were significant
differences observed in the Ct-values during the DNA amplifications using the Fast PCR
cycle conditions.
The Ct-values obtained in the Standard PCR cycle conditions for wild type Taq enzyme
was ~16 cycles while the same for the instant mutant Taq enzyme was ~14 cycles. The
Ct-values obtained in the Fast PCR cycle conditions for wild type Taq enzyme was ~39
cycles while the same for the mutant enzyme was ~23 cycles. The differences in Ctvalues
are very significant in terms of processivity and indicate that the instant mutant
Taq enzyme performed faster than the wild enzyme both under Standard and Fast PCR
cycle conditions generating 4-times and ~65,000-times more amplicons than the wild
type enzyme respectively within the time points measured. However, as the components /
precursors mostly get exhausted by the 30th cycle or so, amounts of amplicons at the end
of PCR would not be significantly different. The above aspects are depicted in figure 8,
as Real-Time PCR Data, and the corresponding Gel Electrophoresis analysis is depicted
in figure 9.
EXAMPLE 11:
Instant Taq polymerase in combination with other modifying enzymes: Amplification of
~ 600 bp Viral Coat protein cDNA from Plant Samples by single tube Reverse
Transcriptase reaction
Total RNA was extracted from Plant sample. The cDNA of Viral coat protein was
prepared from the extracted Total RNA and amplified in a single tube Reverse
Transcriptase reaction. This single tube reaction consisted of the instant Taq Polymerase
along with Reverse Transcriptases like MMuLV or AMV, and the amplification was
done under fast PCR reaction condition.
26
Fast PCR Cycle Conditions followed for cDNA amplification are provided in the table 24
below:
Table 24
42ºC 94 ºC 94 ºC 55 ºC 72 ºC 72 ºC 4 ºC
30 min 15 min 5 sec 5 sec 1 sec 1 min ∞
30 cycles
Sequence specific primers were used for the amplification of cDNA from plant varieties.
PCR amplicons were observed within the short duration of fast PCR conditions. The
cDNA was successfully amplified using the aforementioned single tube reaction mixture
containing instant Taq DNA Polymerase in combination with other modifying enzymes
like Reverse Trancriptases.
• BENEFITS OF THE PRESENT INVENTION FOR INDUSTRIAL USAGE:
1. Molecular diagnostic applications: Fast protocols for DNA/ RNA isolations within 15
minutes are available. Combined with that, the Fast detection of virus / bacteria can
be completed in pathological samples within 30-45 minutes on a Real-time PCR. This
would make on-site diagnosis possible while the patient waits for the results.
2. Increased efficiency in machine-usage; Upto 5 times more runs per day per machine
for both Standard PCR and Real-Time PCR machines.
3. For high-throughput screening of mutations / SNPs, Fast PCR format will help reduce
time and cost and also increases the screening efficiencies by 3- fold.
4. On-site disease Detection based on Fast PCR protocols.
ADVANTAGES OF FAST PCR FOR CRUDE AND DIFFICULT TEMPLATES
PCR-based diagnosis often requires templates obtained from crude sources like blood,
sputum, stool, urine, etc., which may contain impurities and PCR inhibitors. Hence, it is
of great advantage that the Fast PCR protocols are performed similarly to the standard
PCR protocol. The Fast PCR protocol does not require any specific purification protocol
and can amplify DNA crude samples with the same efficiency as the purified DNA
samples. The amount of amplicons produced in both the cases was also similar and the
27
amplicons could be cloned with similar efficiency. Further, the mutant Taq Polymerase
can be used for PCR amplification of DNA from the following various samples/sources:
Plant: any plant part like the seed, stem, root, flower, leaf etc.
Animal: any part like blood, tissue, hair etc.
Bacteria, Fungi, Algae, Virus etc.
SOME OF THE ADVANTAGES OVER CONVENTIONAL PCR
1. Routine PCRs that requires two and half hours to three hrs can be completed within
40 minutes.
2. The instant mutant Taq enzyme can amplify long-DNA fragments of 5-7 kb within 2
hours.
3. Yield of amplicons for long-PCR is higher by ~50%.
4. Crude samples like bacterial Colony, blood, Soil can be used directly for Fast PCR.
5. Molecular diagnostic applications for detection of virus / bacteria can be completed
within 10-15 min on a Real-time PCR.
The advantages mentioned above are achieved without compromising the following
benefits of the wild-type Taq Polymerase enzyme:
1. The Fidelity of the Taq Polymerase enzyme remains unchanged.
2. The yield of DNA received at the end of routine PCRs remain unchanged, upto 5 μg
from a 50 micro lit reaction volume.
3. The PCR products can be cloned into T/A Cloning vectors with similar success rates.
Special Features of instant Mutated Taq Polymerase:
1. The instant enzyme can be used in combination with other enzymes like Reverse
transcriptase for performing reverse transcriptase-PCR (RT-PCR) reactions.
2. The instant enzyme can also be used in combination with proof-reading polymerase
for performing long PCRs.
3. The instant enzyme can also be used for DNA Fingerprinting experiments like
RAPD, RFLP, ARMS-PCR etc.
28
The properties of the instant mutated Taq Polymerase vis-à-vis the wild type Taq
Polymerase is provided in the below table 24:
Table 24
DNA
Polymerase
Biological
Source
5'--3'
Exonuclease
Activity
3'--5'
Exonuclease
Activity
95°C
Halflife
(min)
Commercial
Names
Extension Rate
(nucleotides/s) Error Rate
Time
(s) to
1kb
(72°C)
Taq
polymerase
(Wild type)
Thermus
Aquaticus + - 40 Taq
polymearse 35-100
66 mutations
in 10^6
bases
incorporated
60
MutatedTaq Thermus
Aquaticus + - 40 FasTaqTM
polymearse 500
66 mutations
in 10^6
bases
incorporated
2
It can be observed from the above table that the instant mutant Taq polymerase enzyme
has similar properties vis-a-vis the wild-type Taq polymerase enzyme. Both the enzymes
have similar 5' to 3' Exonuclease Activity and fidelity but differ significantly with respect
to their levels of processivity. The instant modified Taq polymerase enzyme with its
mutation due to a nucleotide substitution at the 446th amino acid residue has enhanced
processivity in comparison with the wild-type Taq polymerase enzyme. Hence, the total
duration of time required for the completion of standard PCR amplification protocols
using the instant mutant enzyme is significantly lower than duration observed with the
wild-type Taq polymerase enzyme.
29
SEQUENCE LISTING
<110> CHROMOUS BIOTECH PVT. LTD
<120> A POLYNUCLEOTIDE, POLYPEPTIDE SEQUENCE AND METHODS
THEREOF
<130> IP13398
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2499
<212> DNA
<213> Thermus aquaticus
<220>
<221> gene
<222> (1)..(2499)
<400> 1
atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60
cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120
gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180
gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240
tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300
gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360
gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420
30
gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480
tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540
gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600
gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660
ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720
ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780
aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840
ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900
ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960
cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020
gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080
ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140
gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200
gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260
gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320
ctggcccaca tggaggtcac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380
31
ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440
cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500
cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560
gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620
ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680
cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740
ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800
gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860
cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920
gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980
gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040
gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100
cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160
gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220
cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280
32
atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340
cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400
cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460
gtggggatag gggaggactg gctctccgcc aaggagtga 2499
<210> 2
<211> 832
<212> PRT
<213> Thermus aquaticus
<220>
<221> PEPTIDE
<222> (1)..(832)
<400> 2
Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu
1 5 10 15
Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly
20 25 30
Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val
50 55 60
Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly
65 70 75 80
Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95
Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110
33
Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys
115 120 125
Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp
130 135 140
Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly
145 150 155 160
Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175
Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn
180 185 190
Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu
195 200 205
Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu
210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys
225 230 235 240
Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255
Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu
275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly
290 295 300
Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp
305 310 315 320
Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro
325 330 335
Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu
340 345 350
34
Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro
355 360 365
Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn
370 375 380
Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu
385 390 395 400
Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415
Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu
420 425 430
Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Val Thr Gly
435 440 445
Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala
450 455 460
Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His
465 470 475 480
Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp
485 490 495
Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg
500 505 510
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile
515 520 525
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
530 535 540
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu
545 550 555 560
His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575
Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln
580 585 590
35
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605
Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly
610 615 620
Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr
625 630 635 640
Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655
Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670
Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685
Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg
690 695 700
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val
705 710 715 720
Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735
Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro
740 745 750
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu
755 760 765
Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala
785 790 795 800
Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815
Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
36
<210> 3
<211> 20
<212> DNA
<213> Thermus aquaticus
<220>
<221> primer_bind
<222> (1)..(20)
<400> 3
tccgagaggc tcttcgccaa 20
<210> 4
<211> 20
<212> DNA
<213> Thermus aquaticus
<220>
<221> primer_bind
<222> (1)..(20)
<400> 4
taggccacgt ccaggcgcac 20
37
We claim
1. A nucleotide sequence encoding DNA polymerase as set forth in Sequence Id No.
1.
2. The nucleotide sequence as claimed in claim 1, wherein the DNA polymerase is
mutated Taq DNA polymerase.
3. The nucleotide sequence as claimed in claim 2, wherein the mutation is a
nucleotide substitution at 1337th position.
4. A polypeptide sequence as set forth in Sequence Id No. 2.
5. The polypeptide sequence as claimed in claim 4, wherein the polypeptide
sequence is a mutated Taq DNA polymerase polypeptide.
6. The polypeptide sequence as claimed in claim 5, wherein the polypeptide
sequence comprises a mutation at 446th amino acid residue.
7. The polypeptide sequence as claimed in claim 4, wherein the polypeptide
sequence corresponds to nucleotide sequence set forth in Sequence Id No. 1.
8. A genetically modified DNA polymerase comprising polypeptide sequence as set
forth in Sequence Id No. 2 along with storage buffer.
9. The genetically modified DNA Polymerase as claimed in claim 8, wherein the
storage buffer is Taq Storage buffer comprising Tris, KCl, EDTA, Tween20, NP-
40, DTT and Glycerol.
10. The genetically modified DNA Polymerase as claimed in claim 9, wherein the
Taq storage buffer preferably comprises about 50mM Tris at pH 8.0, about 50mM
KCl, about 1mM EDTA, about 0.5% Tween20, about 0.5% NP-40, about 1mM
DTT and about 50% Glycerol.
11. The genetically modified DNA Polymerase as claimed in claim 8, wherein the
DNA polymerase is a Taq DNA polymerase.
12. The genetically modified DNA polymerase as claimed in claim 8, wherein the
polypeptide sequence corresponds to nucleotide sequence set forth in Sequence Id
No. 1.
13. The genetically modified DNA Polymerase as claimed in claim 10, wherein the
polypeptide sequence comprises a mutation at the 446th amino acid residue and
the nucleotide sequence has a nucleotide substitution at 1337th position.
38
14. The genetically modified DNA polymerase as claimed in claim 8, wherein the
DNA polymerase has processivity of amplifying about 500 base pairs per second.
15. The genetically modified DNA polymerase as claimed in claim 8, wherein the
DNA polymerase can be used in combination with other modifying enzymes
selected from a group comprising Reverse Transcriptases selected from a group
consisting AMV-RT and MMuLV-RT, and Proof-reading Polymerase preferably
Pfu Polymerase.
16. A recombinant vector comprising nucleotide sequence set forth in Sequence Id
No.1.
17. The recombinant vector as claimed in claim 16, wherein the vector is pUC 18.
18. The recombinant vector as claimed in claim 16, wherein the nucleotide sequence
encodes a DNA polymerase having a nucleotide substitution at 1337th position.
19. A recombinant host cell, transformed by introduction of a vector comprising
nucleotide sequence set forth in Sequence Id No.1.
20. The recombinant host cell as claimed in claim 19, wherein the host cell is selected
from a group comprising E. coli JM series, E. coli DH series and E. coli with lac
operon system, and wherein the vector is pUC 18.
21. The vector as claimed in claim 19, wherein the nucleotide sequence encodes a
DNA polymerase having a nucleotide substitution at 1337th position.
22. Primers as set forth in Sequence Id No. 3 and Sequence Id No 4.
23. The primer as claimed in claim 22, wherein the primer set forth in Sequence Id
No 3 is forward primer and the primer set forth in Sequence Id No 4 is reverse
primer.
24. The primer as claimed in claim 22, wherein the primer is used for introducing
mutations in DNA polymerase by site directed random mutagenesis.
25. A method of amplification of nucleotide sequences, said method comprising step
of amplifying the nucleotide sequence by a DNA polymerase as set forth in
Sequence Id No.1.
26. The method as claimed in claim 25, wherein the DNA polymerase is a mutated
Taq DNA polymerase having a nucleotide substitution at 1337th position.
39
27. The method as claimed in claim 25, wherein the DNA polymerase has
processivity of amplifying about 500 base pairs per second.
28. A PCR reaction mixture, said mixture comprising sample to be detected, probes,
primers, DNA polymerase comprising the nucleotide sequence set forth in
Sequence Id No. 1 and nucleic acid amplification reagents.
29. The PCR reaction mixture as claimed in claim 28, wherein the sample comprises
genetic material selected from a group comprising Plants, Animals, Bacteria,
Fungi, Algae and Virus.
30. The PCR reaction mixture as claimed in claim 28, wherein the DNA polymerase
is a mutated Taq DNA polymerase having a nucleotide substitution at 1337th
position.
31. The PCR reaction mixture as claimed in claim 28, wherein the DNA polymerase
has processivity of amplifying about 500 base pairs per second.
32. A kit comprising probes, primers, DNA polymerase having nucleotide sequence
set forth in Sequence Id No.1 or polypeptide sequence set forth in Sequence Id
No.2 and amplification reagents.
33. The kit as claimed in claim 32, wherein the DNA polymerase is a mutated Taq
DNA polymerase having a nucleotide substitution at 1337th position.
34. The kit as claimed in claim 32, wherein the DNA polymerase has enhanced
processivity of amplifying about 500 base pairs per second.
35. The kit as claimed in claim 32, wherein the kit is used for performing PCR
amplifications.

Dated this 07th day of May, 2010

Signature:
Name: Durgesh Mukharya

To Of K & S Partners
The Controller of Patent Agent for the Applicant
The Patent Office, at Chennai