U.S. patent application number 13/799800 was filed with the patent office on 2014-04-24 for direct nucleic acid amplification kit, reagent and method.
This patent application is currently assigned to GE HEALTHCARE UK LIMITED. The applicant listed for this patent is GE HEALTHCARE UK LIMITED. Invention is credited to JEFFREY K. HORTON, KATHRYN L. LAMERTON, PETER J. TATNELL.
Application Number | 20140113294 13/799800 |
Document ID | / |
Family ID | 47359421 |
Filed Date | 2014-04-24 |
United States Patent
Application |
20140113294 |
Kind Code |
A1 |
HORTON; JEFFREY K. ; et
al. |
April 24, 2014 |
DIRECT NUCLEIC ACID AMPLIFICATION KIT, REAGENT AND METHOD
Abstract
The present invention relates to compositions, methods and kits
which can be used to amplify nucleic acids with the advantage of
decreasing user time and possible contamination. The dried reagent
composition of the invention can be used for easy processing and
amplification of nucleic acid samples.
Inventors: |
HORTON; JEFFREY K.;
(CARDIFF, GB) ; TATNELL; PETER J.; (CARDIFF,
GB) ; LAMERTON; KATHRYN L.; (CARDIFF, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GE HEALTHCARE UK LIMITED |
Little Chalfont |
|
GB |
|
|
Assignee: |
GE HEALTHCARE UK LIMITED
LITTLE CHALFONT
GB
|
Family ID: |
47359421 |
Appl. No.: |
13/799800 |
Filed: |
March 13, 2013 |
Current U.S.
Class: |
435/6.12 ;
435/194; 435/91.2 |
Current CPC
Class: |
C12Q 1/686 20130101;
C12P 19/34 20130101; C12Q 2527/125 20130101; C12Q 1/686 20130101;
C12Q 2527/125 20130101 |
Class at
Publication: |
435/6.12 ;
435/194; 435/91.2 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 24, 2012 |
GB |
1219137.5 |
Claims
1. A dried reagent composition for nucleic acid amplification
comprising a sequestering reagent, a polymerase and a
deoxyribonucleotide triphosphate (dNTP).
2. The dried reagent composition of claim 1, wherein said
sequestering agent is a cyclodextrin.
3. The dried reagent composition of claim 2, wherein said
cyclodextrin is selected from a group consisting of
.alpha.-cyclodextrin, .beta.-cyclodextrin, .gamma.-cyclodextrin,
6-O-.alpha.-D-Maltosyl-.beta. cyclodextrin,
hydroxyethyl-.beta.-cyclodextrin, hydroxypropyl-.beta.-cyclodextrin
and 2-hydroxypropyl-.beta.-cyclodextrin and derivatives
thereof.
4. The dried reagent composition of claim 2, wherein the
cyclodextrin is .alpha.-cyclodextrin.
5. The dried reagent composition of claim 1, further comprising at
least one primer.
6. The dried reagent composition of claim 1, further comprising an
excipient mix.
7. The dried reagent composition of claim 6, wherein the excipient
mix comprises 10.times.PCR buffer, Ficoll 70, Ficoll 400,
Melezitose and nuclease free water.
8. The dried reagent composition of claim 1, further comprising an
exchange buffer.
9. The dried reagent composition of claim 1, further comprising
bovine serum albumin (BSA).
10. The dried reagent composition of claim 1, wherein said
polymerase is OmniKlen Taq (OKT).
11. The dried reagent composition of claim 1, comprising
.alpha.-cyclodextrin, at least one primer, a polymerase, dNTP, BSA,
an excipient mix and an exchange buffer.
12. A method for producing a dried reagent composition for nucleic
acid amplification comprising the steps: i) combining a polymerase
with a sequestering reagent and dNTP, to provide a mixture thereof;
and ii) drying said mixture to form a dried reagent
composition.
13. The method of claim 12, wherein the mixture of step i) further
comprises at least one primer.
14. The method of claim 12, wherein the mixture of step i) further
comprises an excipient mix.
15. The method of claim 12, wherein the mixture of step i) further
comprises an exchange buffer.
16. The method of claim 12, wherein said drying step is achieved by
lyophilizing.
17. The method of claim 12, further comprising freezing said
composition prior to said drying step.
18. A method for amplification of nucleic acid comprising the
steps: i) incubating a solution containing nucleic acid with the
dried reagent composition of claim 1; and ii) amplifying said
nucleic acid.
19. The nucleic acid amplification method of claim 18, wherein the
amplification method is a polymerase chain reaction.
20. The nucleic acid amplification method of claim 18, wherein the
amplification method comprises reverse transcription polymerase
chain reaction or isothermal amplification.
21. The method of claim 18, further comprising a step prior to step
i) wherein said solution is formed by adding water to the nucleic
acid.
22. The method of claim 18, wherein the nucleic acid is immobilised
on a solid support.
23. The method of claim 22, wherein said solid support is a
cellulose based matrix.
24. The method of claim 22, wherein the solid support is in the
form of a pre punched disc.
25. The method of claim 22, wherein the solid support is in the
form of an FTA pre punched disc.
26. The method of claim 22, wherein a lysis reagent is embedded
onto said solid matrix.
27. The method of claim 18, wherein the amplification is carried
out in a single reaction vessel.
28. A method of detecting and/or quantifying amplified nucleic acid
using a detection system comprising the steps: i) amplifying
nucleic acids using the method of claim 18 to produce amplified
nucleic acid; ii) detecting said amplified nucleic acid; and iii)
optionally quantifying the amplified nucleic acid.
29. The method of claim 28, wherein said detection system is a PCR
imaging system.
30. A kit for amplifying the nucleic acid comprising the dried
reagent of claim 1 and instructions for use thereof.
Description
FIELD OF INVENTION
[0001] The present invention relates to the field of nucleic acid
amplification, particularly to the use of a polymerase chain
reaction to amplify nucleic acids. The invention provides methods
and kits which can be used to amplify nucleic acids by lyophilizing
or freeze-drying nucleic acid amplification reagents for easy
amplification of nucleic acid samples. The invention has
applications in the easy processing of nucleic acids and is
particularly useful in genotyping, diagnostics and forensics.
BACKGROUND OF THE INVENTION
[0002] The polymerase chain reaction (PCR) is a common tool used in
molecular biology for amplifying nucleic acids. U.S. Pat. No.
4,683,202 (Mullis, Cetus Corporation) describes a process for
amplifying any desired specific nucleic acid sequence contained in
a nucleic acid or mixture thereof.
[0003] U.S. Pat. No. 5,705,345 (Lundin et al.) describes a method
of nucleic acid preparation whereby the sample containing cells is
lysed to release nucleic acid and the sample is treated with
cyclodextrin to neutralize the extractant. The advantage of this
system is that conventional detergent removal requires a separation
step; however the addition of cyclodextrin to neutralize the
detergent removes the need for the separation step and thus reduces
the risk of contamination.
[0004] WO 99/38962 (Health, Gentra Systems Inc.) describes a solid
support with a bound lysis reagent. The lysis reagent can comprise
a detergent, a chelating agent, water and optionally an RNA
digesting enzyme. The method for PCR amplification requires further
steps for purification of the nucleic acid for amplification
analysis.
[0005] WO 91/02040 (Kosak) describes an invention using
cyclodextrin-labelled primers in an amplification reaction mixture
for qualitative and quantitative nucleic acid sequence analysis.
The benefits of this invention are a higher signal efficiency and
versatility in label colours.
[0006] WO 95/32739 (Agrawal) describes an oligonucleotide
non-covalently complexed with a cyclodextrin. However the
incorporation of cyclodextrin with oligonucleotides was for the
cellular uptake of oligonucleotides and not for the amplification
of nucleotides in a PCR reaction.
[0007] WO 2010/066908 (Beckers et al.,) describes the use of
cyclodextrins to improve the specificity, sensitivity and/or yield
of PCR. The method discloses an amplification reaction which is
performed in a reaction mixture comprising at least one
cyclodextrin and performing the amplification reaction on the
reaction mixture. Again the advantage being a combination of
cyclodextrin in the liquid amplification reaction mixture would
reduce the risk of contamination by reducing the number of
purification steps.
[0008] Current methods for DNA amplification involve a DNA
purification procedure which often involves several steps which
increases the chance of contamination. This is a tedious process
and prior art methods have a number of clear disadvantages in terms
of cost, complexity and in particular, user time. For example,
column-based nucleic acid purification is a typical solid phase
extraction method to purify nucleic acids. This method relies on
the nucleic acid binding through adsorption to silica or other
supports depending on the pH and the salt content of the buffer.
Examples of suitable buffers include Tris-EDTA (TE) buffer or
Phosphate buffer (used in DNA microarray experiments due to the
reactive amines). The purification of nucleic acids on such spin
columns includes a number of complex and tedious steps. Nucleic
acid purification on spin columns typically involves three
time-consuming and complex steps/stages: the sample containing
nucleic acid is added to the column and the nucleic acid binds due
to the lower pH (relative to the silanol groups on the column) and
salt concentration of the binding solution, which may contain
buffer, a denaturing agent (such as guanidine hydrochloride),
Triton X-100, isopropanol and a pH indicator; the column is washed
with 5 mM KPO.sub.4 pH 8.0 or similar, 80% EtOH); and the column is
eluted with buffer or water.
[0009] Alternative methods involve the binding of nucleic acids in
the presence of chaotropic agents such that DNA binds to silica or
glass particles or glass beads. This property was used to purify
nucleic acid using glass powder or silica beads under alkaline
conditions. Typical chaotropic agents include guanidinium
thiocyanate or guanidinium hydrochloride and recently glass beads
have been substituted with glass containing minicolumns.
[0010] Some of the pitfalls of quantitative real-time reverse
transcription polymerase chain reaction, including the effect of
inhibitors, are described by Bustin & Nolan (J. Biomolecular
Techniques, 2004, 15, 155-166).
[0011] The best defense against PCR amplification failure in
forensics applications is to combine sound sample handling and
processing techniques with extraction systems proven to efficiently
purify DNA.
[0012] Typically PCR reagents are stored in glycerol solution which
must be maintained at temperatures below room temperature.
Lyophilisation or freeze drying is a process widely used in the
preparation of reagents for nucleic acid analysis and other
biological processes because it allows for long term stability of
otherwise labile biomolecules, and provides a convenient method of
storage, shipping and reconstitution. However there are numerous
technical challenges involved in producing a lyophilised or
freeze-dried reagent for PCR analysis. Current technology for
producing dry biological reagent compositions involves procedures
such as dry-blending, spray-drying, freeze-drying, fluidized bed
drying, and/or cryogenic freezing. All of these procedures have
limitations and drawbacks including consistency and
reliability.
[0013] With dry-blending technology (Muzzio et al 2002, Powder
Technology 124, 1-7), it is often difficult to obtain homogeneous
blends of chemicals due to their different densities. Furthermore,
homogeneity is especially difficult to achieve when very small
amounts of ingredients are mixed with large amounts of other
ingredients. Even if homogeneity is achieved, it is difficult to
reproducibly dispense small amounts of the blended biological
chemicals.
[0014] Spray-drying technology (U.S. Pat. No. 4,712,310) provides
more homogeneous blends of chemicals because the reagents are first
dissolved in solution. With spray-drying, however, it is difficult
to dispense precise amounts of blended chemicals. To overcome this
drawback, the resulting particles are usually reprocessed by
agglomeration to obtain uniform particle sizes such as tablets.
However, the agglomerated particles are generally less soluble than
the original spray-dried particles or powders. Also, these
procedures sometimes use fluorocarbon cryogenic solutions which can
be hazardous to the environment.
[0015] Fluid bed technology (Rubina, 1999, Pharmaceutical
Technology, 23, 104-113) relies upon spraying a liquid reagent
blend onto a particle and drying the liquid to obtain a particle
coated with the blended reagents. Using this procedure, it is
difficult to obtain uniformly sized particles and to produce a
uniform coating.
[0016] Another method for stabilizing biologics is freeze-drying.
One drawback to the freeze-drying is the use of fluorocarbon
refrigerants which are difficult to dispose of and the
freeze-drying process may be imprecise and also difficult to
regulate. Indeed, regular freeze drying of reagents may not provide
an entire solution for particularly labile reagents used in
molecular biology processes. Furthermore, degradation of the
product during the freeze drying process is common and a freeze
dried product is not always perfectly stable during storage.
Process control is critical and can be difficult to regulate (Tang
& Pakil, 2004, Design of Freeze-Drying Processes for
Pharmaceuticals: Practical Advice; Pharmaceutical Research, 21,
191-200).
[0017] Another method of stabilizing biologics is by air-drying
biological reagent compositions (Ratti, 2001, Hot Air and
Freeze-Drying of High Value Foods: A Review. J. Food Engineering
49, 311-319). Some problems with air drying processes are that the
dried product is not in a readily dispensable form. Also, the
biological reagents must be stable at or above the temperature of
the drying process and it is a difficult process to control
accurately.
[0018] One specialized process using freeze-drying technology is
the formation of droplets or spheres which are contacted with a
cryogenic liquid and then freeze-dried. One drawback of this
technology is that the reagent spheres are fragile and tend to
disintegrate.
[0019] One type of carrier or filler which has been used to
stabilize biological reagents are glass-forming filler materials
(U.S. Pat. No. 5,565,318). The biological reagent solutions are
incorporated into the glass-forming filler materials (which are
water soluble or a water-swellable substance). They are then dried
to produce a glassy composition which immobilizes and stabilizes
the biological reagent (U.S. Pat. No. 5,593,824).
[0020] Carbohydrates such as glucose, sucrose, maltose or
maltotriose are an important group of glass-forming substances.
Other polyhydroxy compounds can be used such as carbohydrate
derivatives like sorbitol and chemically modified carbohydrates.
Another important class of glass-forming substances are synthetic
polymers such as polyvinyl pyrrolidone, polyacrylamide, or
polyethyleneimine.
[0021] Further examples of glass-forming substances include sugar
copolymers such as Ficoll.TM. (U.S. Pat. No. 3,300,474). Ficoll is
a neutral, highly branched, high-mass, hydrophilic polysaccharide
which dissolves readily in aqueous solutions. Ficoll radii range
from 2-7 nm. It is prepared by reaction of the polysaccharide with
epichlorohydrin. Ficoll has molecular weights of between 5,000 to
1,000,000 and contains sucrose residues linked through ether
bridges to bifunctional groups. Such groups may be an alkylene of
2, 3 or more carbon atoms but not normally more than 10 carbon
atoms. The bifunctional groups serve to connect sugar residues
together. These polymers may, for example, be made by the reaction
of sugar with a halohydrin or bis-epoxy compound. A glass is
typically defined as an undercooled liquid with a very high
viscosity,
[0022] One drawback of the aforementioned references is that
normally the stabilized and glassified biological materials are
ground into powders, compounded into tablets, or maintained in a
thin glassy film in a container like a microtitre plate. Numerous
methods to make and use compositions of glassy immobilized
biological materials have been tried. One system utilizes a thin
glassy film dried and dispensed into a container suitable to the
final user, such as a micro centrifuge tube. However, this process
has resulted only in limited success.
[0023] There is therefore a need for an improved and simplified
process for performing polymerase chain reaction from samples prior
to nucleic acid amplification by PCR. Furthermore, there is a need
for PCR reagent mixtures which can be stored, shipped and
reconstituted at room temperature without the need for storage in
glycerol and lower temperatures. The present invention addresses
these problems and provides methods and kits which can be used for
single step amplification of nucleic acid.
SUMMARY OF THE INVENTION
[0024] The present invention provides methods and kits which can be
used to amplify nucleic acids by incorporating all the required PCR
reagents into a lyophilized format for easy amplification of
nucleic acid samples.
[0025] According to a first aspect of the present invention there
is provided a dried reagent composition for nucleic acid
amplification comprising, a sequestering reagent, a polymerase and
a deoxyribonucleotide triphosphate (dNTP). The advantage of
incorporating the sequestrant with the polymerase and dNTP into a
dried reagent composition is to reduce the number of steps required
for nucleic acid amplification, thus saving operator time and
facilitating operator usage.
[0026] In one aspect, the nucleic acid is selected from the group
consisting of DNA, RNA and oligonucleotide.
[0027] The term "nucleic acid" is used herein synonymously with the
term "nucleotides" and includes DNA, such as plasmid DNA and
genomic DNA; RNA, such as mRNA, tRNA, sRNA and RNAi; and protein
nucleic acid, PNA.
[0028] In another aspect, the sequestering agent is a cyclodextrin.
The cyclodextrin may be selected from a group consisting of
.alpha.-cyclodextrin, .beta.-cyclodextrin, .gamma.-cyclodextrin and
derivatives thereof. Cyclodextrin could consist of a group
consisting of 6-O-.alpha.-D-Maltosyl-.beta. cyclodextrin,
hydroxyethyl-.beta.-cyclodextrin, hydroxypropyl-.beta.-cyclodextrin
and 2-hydroxypropyl-.beta.-cyclodextrin. The sequestrant is
preferably .alpha.-cyclodextrin. The sequestering reagent is not a
chelating agent. A chelating agent is a chemical compound that
combines with a metal to form a chelate, often used to trap heavy
metal ions (Colins English Dictionary, .COPYRGT. HarperCollins
Publishers 2003). One example of a lysis reagent is sodium dodecyl
suphate; sodium is a metal ion however according to Ramamurthy
Palepu and Vincent C. Reinsborough (Can J. Chem Vol 66, 325-328,
1988) it is the hydrophobic tail that interacts with the
cyclodextrin not the hydrophilic head.
[0029] In a further aspect, the dried reagent composition comprises
at least one primer.
[0030] In a further aspect, the dried reagent composition
additionally comprises an excipient mix.
[0031] The term "excipient mix" is used herein to denote additives
or ingredients used to make up a preparation or mixture and for
example may comprise of PCR buffer, Ficoll 70, Ficoll 400,
Melezitose, Trehalose, stabilising proteins and nuclease free
water.
[0032] The term "PCR buffer" is used herein to denote a buffer
necessary to create optimal conditions for activity of a DNA
polymerase and for example may comprise of Tris-HCl, KCl,
MgCl.sub.2, gelatin and nuclease free water.
[0033] In a further aspect, the dried reagent composition
additionally comprises an exchange buffer.
[0034] The term "exchange buffer" is used herein to denote a buffer
used for the removal of small ionic solutes, whereby one buffer is
removed and replaced with another alternative buffer and for
example may comprise of Tris/HCl, CaCl.sub.2, a detergent, RE960,
MgCl.sub.2, KCl and nuclease free water.
[0035] In a further aspect, the dried reagent composition
additionally comprises bovine serum albumin (BSA).
[0036] In one aspect, the polymerase is an OmniKlen Taq (OKT)
Polymerase. Alternatively, the polymerase may be selected from the
group consisting of T4 DNA Polymerase, Pol I and Klenow Fragment,
T4 DNA Polymerase, Modified Bacteriophage T7 DNA Polymerase,
Terminal Deoxynucleotide Transferase, Bst Polymerase, Taq
Polymerase, Tth polymerase, Pow Polymerase, Vent Polymerase, Pab
Pol I DNA Polymerase, Thermus thermophiles, Carboxydothermus
hydrogenoformans, SP6 and SP7 RNA polymerase.
[0037] In a further aspect, the preferred embodiment is a dried
reagent composition comprising .alpha.-cyclodextrin, at least one
primer, a polymerase, dNTP, BSA, an excipient mix and an exchange
buffer.
[0038] According to a second aspect of the present invention, there
is provided a method for producing a dried reagent composition for
nucleic acid amplification comprising the steps: [0039] i)
combining a polymerase with a sequestering reagent and dNTP, to
provide a mixture thereof, and [0040] ii) drying said mixture to
form a dried reagent composition.
[0041] In one aspect, the mixture additionally comprises at least
one primer.
[0042] In another aspect, the mixture additionally comprises an
excipient mix.
[0043] In a further aspect, the mixture additionally comprises an
exchange buffer.
[0044] In one aspect, the drying step is achieved by lyophilizing
the mixture.
[0045] In another aspect, the additionally comprises the step of
freezing the composition prior to the drying step.
[0046] According to a third aspect of the present invention, there
is provided a method for amplification of nucleic acid comprising
the steps: [0047] i) incubating a solution containing nucleic acid
with the dried reagent composition described above; and [0048] ii)
amplifying the nucleic acid.
[0049] In one aspect, the amplification method is a polymerase
chain reaction.
[0050] In another aspect, the amplification method comprises
reverse transcription polymerase chain reaction or isothermal
amplification.
[0051] In a further aspect, prior to step i), the solution is
formed by adding water to the nucleic acid.
[0052] In one aspect, the nucleic acid is immobilized on a solid
support.
[0053] In another aspect, the solid support is a cellulose based
matrix.
[0054] In a further aspect, the solid matrix is selected from the
group consisting of glass, glass fiber, glass microfiber, silica,
silica gel, silica oxide, cellulose, nitrocellulose,
carboxymethylcellulose, polyester, polyamide, carbohydrate
polymers, polypropylene, polytetraflurorethylene,
polyvinylidinefluoride, wool and porous ceramics. The solid matrix
may comprise a glass or silica-based solid phase medium, a
plastics-based solid phase medium or a cellulose-based solid phase
medium. The solid support is preferably a cellulose-based matrix.
Examples of cellulose-based matrices include FTA.TM. (data file
51668), 903 neonatal cards and 31-ETF cards available from GE
Healthcare.
[0055] In one aspect, the cellulose based matrix is in the form of
a pre punched disc.
[0056] In another aspect, the cellulose based matrix is in the form
of an FTA pre punched disc.
[0057] In a further aspect, the lysis reagent is embedded onto said
solid matrix.
[0058] In one aspect, the lysis reagent comprises an anionic
surfactant or detergent. Sodium dodecyl sulphate (SDS) is an
example of an anionic surfactant frequently used to lyse biological
cells.
[0059] In another aspect, the amplification is carried out in a
single reaction vessel such as a test tube or the well of a
multi-well plate.
[0060] The method of the invention can be used either in a single
reaction well or a high-throughput 96-well format in combination
with automated sample processing as described by Baron et al.,
(2011, Forensics Science International: Genetics Supplement Series,
93, e560-e561). This approach would involve a minimal number of
steps and increase sample throughput. The risk of operator-induced
error, such as cross-contamination is also reduced since this
procedure requires fewer manipulations compared to protocols
associated with currently used, more labor intensive kits (e.g.
QIAmp DNA blood mini kit, Qiagen). The risk of sample mix-up is
also reduced since the procedure requires few manipulations.
Importantly, the method is readily transferable to a multi-well
format for high-throughput screening. The present invention can
thus improve sample processing for carrying out PCR reactions to
aid genetic interrogations. The invention can be conducted in a 96
well/high throughput format to facilitate sample handling and thus
eliminate batch processing of samples.
[0061] The advantage of dried or lyophilized formulations of the
polymerase chain reaction reagents is that they can be easily
solubilized by the addition of water, thus saving operator time and
facilitating operator usage. To minimize operator error, the dried
reagent mixture can be pre-dispensed into the reaction vessel, such
as the well of a multi-well plate. The preformulated, predispensed,
ambient-temperature-stable beads or cakes allow amplification
reactions to be carried out within a single well or reaction vessel
and ensure greater reproducibility between reactions, minimize
pipetting steps, and reduce the potential for pipetting errors and
contamination.
[0062] According to a fourth aspect of the present invention, there
is provided a method for detecting and/or quantifying amplified
nucleic acid using a detection system comprising the steps: [0063]
i) amplifying nucleic acids using the methods as hereinbefore
described to produce amplified nucleic acid; [0064] ii) detecting
the amplified nucleic acid; and [0065] iii) optionally quantifying
the amplified nucleic acid.
[0066] In one aspect, the detection system is a PCR imaging
system.
[0067] In another aspect, the detection system is a fluorescence or
luminescent based system.
[0068] It will be understood that the nucleic acid may be viral,
prokaryotic or eukaryotic in origin.
[0069] In one, the nucleic acid sample is present in a cellular
sample. The cellular sample may originate from a mammal, bird, fish
or plant or a cell culture thereof. Preferably the cellular sample
is mammalian in origin, most preferably human in origin. The sample
containing the nucleic acid may be derived from any source. This
includes, for example, physiological/pathological body fluids (e.g.
secretions, excretions, exudates) or cell suspensions of humans and
animals; physiological/pathological liquids or cell suspensions of
plants; liquid products, extracts or suspensions of bacteria,
fungi, plasmids, viruses, prions, etc.; liquid extracts or
homogenates of human or animal body tissues (e.g., bone, liver,
kidney, etc.); media from DNA or RNA synthesis, mixtures of
chemically or biochemically synthesized DNA or RNA; and any other
source in which DNA or RNA is or can be in a liquid medium.
[0070] In a further aspect, the method is for use as a tool
selected from the group consisting of a molecular diagnostics tool,
a human identification tool, a forensics tool, STR profiling tool
and DNA profiling.
[0071] According to a fifth aspect of the present invention, there
is provided a kit for amplifying nucleic acid comprising the dried
reagent composition hereinbefore described and instructions for use
thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0072] FIG. 1 shows the results from PCR amplification of unwashed
blood-spotted FTA paper with nucleic acid amplification reagent
cakes with or without .alpha.-cyclodextrin.
[0073] FIG. 2 shows the results from PCR amplification of unwashed
blood-spotted FTA paper with nucleic acid amplification reagent
cakes with .alpha.-cyclodextrin.
[0074] FIG. 3 shows results from PCR amplification of titrated
OmniKlen Taq (OKT) polymerase, in nucleic acid amplification
reagent cakes, demonstrating the best concentration to freeze dry
the nucleic acid amplification formulation.
DETAILED DESCRIPTION OF THE INVENTION
Chemicals and Materials Used
[0075] A list of the chemicals and their sources is given below:
FTA papers for storing nucleic acid were obtained from GE
Healthcare UK Limited; Normal human blood (Tissue Solutions Ltd);
Genomic DNA (Promega product code G152A); 1 kb DNA ladder (Promega
product code G571A); Harris Uni-core punch, 1.2 mm (Sigma,
Catalogue number Z708860-25ea, lot 3110); OmniKlentaq Polymerase
(Mo Bio Inc, catalogue code 1225-250); Deoxyribonucleotide
triphosphate (dNTP) (Life Tech);
PCR Grade Bovine Serum Albumin (Life Tech);
TABLE-US-00001 [0076] Forward and reverse .beta.-globin
primer(Sigma Genosys) (.beta.-globin 1.3 forward
5'-TTAGGCCTTAGCGGGCTTAGAC- 3' (Seq ID No. 1) and .beta.-globin 1.3
reverse 5'- CCAGGATTTTTGATGGGACACG-3' (Seq ID No. 2));
.alpha.-cyclodextrin (Fluka code 28705) and Sterile water (Sigma
Product code W4502).
Excipient Mix:
Ficoll 70 (GE Healthcare);
Ficoll 400 (GE Healthcare) and
Melezitose (Sigma)
Cycle Sequence Mix 10.times.:
Trizma (Sigma);
KCl (Sigma);
MgCl (Sigma) and
[0077] Nuclease-free water (Sigma)
Exchange Buffer:
Tris/HCl pH8.5 (Sigma);
1M CaCl.sub.2 (Sigma);
1.0M MgCl.sub.2 (Sigma);
2.0M KCl (Sigma) and
RHODAFAC RE-960 (7% RE960) (Kao Chemicals)
Experimental Results
[0078] DNA Measurement from Dried Blood Spots from Cellulose
Matrices Using qPCR. PCR reagents were combined with cyclodextrin
and lyophilised under the following conditions in Table 1.
TABLE-US-00002 TABLE 1 Lyophilisation conditions Temperature Vacuum
Time (.degree. C.) (mTorr) (min.) Comment -46 100 600 hold -36 100
250 ramp -36 100 300 hold 0 100 300 hold 28 100 233 ramp 28 100 360
hold Post Heat: 28 100 2000 hold
[0079] Samples were combined with the lyophilised nucleic acid
amplification composition in a 96 well plate.
[0080] PCR reaction was set up as follows:
[0081] Blood-spotted FTA was added to a well with a nucleic acid
amplification reagent cake that contained cyclodextrin or did not
contain cyclodextrin. Standards and samples were added to the
appropriate wells. The plates were centrifuged at 1000 rpm for 1
minute and sealed. PCR was carried out on an MJ Research PTC-200
Thermo Cycler following the manufacturer's user instructions.
[0082] The thermal cycling conditions were: 95.degree. C. for 5
min, 95.degree. C. for 30 sec, 55/65.degree. C. for 1 min,
72.degree. C. for 2 min followed by 35 cycles of: 95.degree. C. for
30 sec, 55/65.degree. C. for 1 min, 72.degree. C. 2 min, followed
by 72.degree. C. for 10 mins.
[0083] Following amplification, visualisation of PCR products was
achieved using agarose gel electrophoresis (1.times.TEA buffer, 1%
agarose gel).
[0084] The standard well of the 96 well PCR plate was loaded with 5
.mu.l of the 1 Kb DNA ladder with 1 .mu.l of 6.times. loading
buffer.
[0085] The results are presented in FIGS. 1, 2 and 3.
[0086] FIG. 1 shows PCR amplification of unwashed blood-spotted FTA
with the lyophilized nucleic acid amplification composition with or
without .alpha.-cyclodextrin: Lane M: 1 kb Ladder; Lane 1-4: FTA
punch spotted with whole blood (1.2 mm) with a lyophilized nucleic
acid amplification composition without cyclodextrin; Lane 5-8: FTA
punch spotted with whole blood (1.2 mm) with the lyophilized
nucleic acid amplification composition containing cyclodextrin.
[0087] FIG. 2 shows PCR amplification of unwashed blood-spotted FTA
with the lyophilized nucleic acid amplification composition with or
without .alpha.-cyclodextrin: Lane M: 1 kb Ladder; Lane 1-2: FTA
punch spotted with whole blood (1.2 mm) with the lyophilized
nucleic acid amplification composition containing cyclodextrin;
Lane 3-4: FTA punch (1.2 mm) with the lyophilized nucleic acid
amplification composition containing cyclodextrin; Lane 5-6: FTA
punch spotted with whole blood (1.2 mm) with the lyophilized
nucleic acid amplification composition without cyclodextrin.
[0088] FIG. 3 shows PCR amplification of unwashed blood-spotted FTA
with the lyophilized nucleic acid amplification composition without
.alpha.-cyclodextrin and with varying concentrations of OKT Taq
polymerase: Lane 1-8 contains 6U OKT, Lane 1-3 FTA punch spotted
with whole blood, Lane 4-6 genomic DNA, Lane 7-8 no DNA template;
Lane 9-16 contains 8U OKT, Lane 9-11 FTA punch spotted with whole
blood, Lane 12-14 genomic DNA, Lane 15-16 no DNA template; Lane
17-24 contains 10U OKT, Lane 17-19 FTA punch spotted with whole
blood, Lane 20-22 genomic DNA, Lane 23-24 no DNA template; Lane
25-32 contains 12U OKT, Lane 25-27 FTA punch spotted with whole
blood, Lane 28-30 genomic DNA, Lane 31-32 no DNA template.
TABLE-US-00003 TABLE 2 Concentration of reagents in the Excipient
mix. Reagent Concentration in 1000 cakes Ficoll 70 0.6933 g Ficoll
400 0.6933 g Melezitose 1.083 g 10x PCR Cycle sequence mix 2.71 ml
10% .alpha.-cyclodextrin 2.855 ml Nuclease-free water 0 ml
TABLE-US-00004 TABLE 3 Concentration of reagents in the Excipient
mix without cyclodextrin. Reagent Concentration in 1000 cakes
Ficoll 70 0.6933 g Ficoll 400 0.6933 g Melezitose 1.083 g Cycle
sequence mix 10x 2.71 ml 10% .alpha.-cyclodextrin 0 ml
Nuclease-free water 2.855 ml
TABLE-US-00005 TABLE 4 Concentration of reagents in a 1000 ml of
10x PCR Cycle Sequence mix. Reagent Concentration in 1000 ml 100 mM
Trizma 12.11 g 500 mm KCl 37.28 g 15 mM MgCl 3.05 g Nuclease-free
water 0.95 L
TABLE-US-00006 TABLE 5 Concentration of reagents in the Exchange
buffer. Reagent Concentration in 1000 ml 1.0M Tris/HCl pH 8.5 20 ml
1.0M CaCl.sub.2 100 ml 7% RE960 214.3 ml 1.0M MgCl.sub.2 2.5 ml
2.0M KCl 16.6 ml Nuclease-free water 746.5 ml
TABLE-US-00007 TABLE 6 Concentration of reagents in the 8U OmniKlen
Taq Polymerase lyophilized nucleic acid amplification composition.
Reagent Concentration in 1000 cakes Excipient Mix 6.667 ml 10 mg/ml
BSA 0.6 ml 20 nM dNTPs 0.25 ml OmniKlen Taq polymerase 0.08 ml
Primer 0.4 pmoles/.mu.l Exchange buffer 0.92 ml Nuclease-free water
1.483 ml
TABLE-US-00008 TABLE 7 Concentration of reagents in the 6U OmniKlen
Taq Polymerase lyophilized nucleic acid amplification composition.
Reagent Concentration in 1000 cakes Excipient Mix without
cyclodextrin 6.667 ml 10 mg/ml BSA 0.6 ml 20 nM dNTPs 0.25 ml
OmniKlen Taq polymerase 0.06 ml Primer 0.4 pmoles/.mu.l Exchange
buffer 0.94 ml Nuclease-free water 1.483 ml
TABLE-US-00009 TABLE 8 Concentration of reagents in the 10U
OmniKlen Taq Polymerase lyophilized nucleic acid amplification
composition. Reagent Concentration in 1000 cakes Excipient Mix
without cyclodextrin 6.667 ml 10 mg/ml BSA 0.6 ml 20 nM dNTPs 0.25
ml OmniKlen Taq polymerase 0.1 ml Primer 0.4 pmoles/.mu.l Exchange
buffer 0.9 ml Nuclease-free water 1.483 ml
TABLE-US-00010 TABLE 9 Concentration of reagents in the 12U
OmniKlen Taq Polymerase lyophilized nucleic acid amplification
composition. Reagent Concentration in 1000 cakes Excipient Mix
without cyclodextrin 6.667 ml 10 mg/ml BSA 0.6 ml 20 nM dNTPs 0.25
ml OmniKlen Taq polymerase 0.12 ml Primer 0.4 pmoles/.mu.l Exchange
buffer 0.92 ml Nuclease-free water 1.443 ml
[0089] While preferred illustrative embodiments of the present
invention are described, one skilled in the art will appreciate
that the present invention can be practiced by other than the
described embodiments, which are presented for the purposes of
illustration only and not by way of limitation. The present
invention is limited only by the claims that follow.
Sequence CWU 1
1
2122DNAArtificial SequencePrimer 1ttaggcctta gcgggcttag ac
22222DNAArtificialPCR primer 2ccaggatttt tgatgggaca cg 22
* * * * *