GB2512631A

GB2512631A - Quantitative detection of specific nucleic acid sequences

Info

Publication number: GB2512631A
Application number: GB1306093.4A
Authority: GB
Inventors: Rupert Maxwell Gaut
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-04-03
Filing date: 2013-04-03
Publication date: 2014-10-08
Also published as: GB201306093D0

Abstract

A PCR reaction mixture for quantitative detection of a specific nucleic acid sequence, comprising a first single-stranded polynucleotide sequence comprising a primer region, a hybridisation region and a fluorophore molecule; a second single-stranded polynucleotide sequence comprising a primer region, a hybridisation region complementary to the hybridisation region of said first single-stranded polynucleotide sequence and a quencher molecule. Hybridisation of the hybridisation region from the first single stranded polynucleotide sequence with the hybridisation region from the second single-stranded polynucleotide sequence brings the fluorophore molecule and quencher molecule into close proximity such that the fluorophore molecule is quenched by the quencher molecule.

Description

Quantitative Detection Qf Specific Nucleic Acid Sequences

CROSS REFERENCE TO RELATED APPLICATIONS

This application represents the first application for a patent directed towards the invention and the subject matter.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a Polymerase Chain Reaction (PCR) reaction mixture and a method for the quantitative detection of a specific nucleic acid sequence. This F'CR reaction mixture and method use high annealing and extension temperatures, which are closer to the optimal conditions for extension of the specific nucleic acid sequence and which result in reduced PCR durations.

2. Description of the Related Art

It is known to detect specific nucleic acid sequences using the Polymerase Chain Reaction (PCR) technique and since the introduction into laboratories in the 1980s of PCR, there have been substantial increases in its speed and sensitivity.

PCR utilizes a pair of short oligonucleotides known as primers, which act to define the region that is to be amplified. Amplification then occurs via fl..

* repeated cycles of a step-wise process, whereby the primer sequences bind to **.* their target sequence and are extended by the action of a polymerase. These cyclical steps lead to amplification in an exponential manner as the amount of target deoxyribonucleic acid (DNA) is doubled after each cycle. *

***.** * . PCR consists of three steps, which are usually distinguished by three different temperatures; a denaturation temperature (95-99°C), an annealing temperature (55-65°C) and an extension temperature (circa 72°C). The exact temperatures, and the duration of each step, are defined by the objective of each step.

Thus, during the denaturation step, the desired outcome is to separate all the double-stranded target sequences present in the sample, so that they may become available for the annealing of primers during the next stage of the PCR process. Annealing temperatures are generally optimized to be 3-5°C below the predicted melting temperature (Tm) of the primers used in the reaction.

Annealing temperatures are required to be sufficiently below the Tm in order to allow efficient binding of the primers to their target sequences but not so low as to pemiit non-specific binding to other sequences present in the target sample.

After the annealing step, the reaction is raised to a temperature which is optimal for the extension of the primer sequences by the DNA polymerase, typically around 72°C. The enzyme Taq polymerase is frequently used as the DNA polymerase.

During the original development of PCR protocols, the post-PCR processing of the reaction was an intrinsic part of the entire procedure and a PCR product was required that was amenable to such procedures as * restriction enzyme digestion and visualization using an ethidium-bromide * * stained agarose gel, as such PCRs were designed to produce relatively large products (generally in excess of a few hundred base pairs, often up to several kilobase pairs). The amplification of large products required relatively long *:::: extension times (>1 minute) and many cycling steps (>40), leading to average PCR times of I to 2 hours. With the introduction of real-time closed-tube PCR techniques utilizing fluorescent dyes, the sensitivity of PCR has increased greatly, requiring fewer cycles before a product can be detected. Also, real-time technologies have greatly reduced the size of the PCR product, or amplicon, required to yield sufficient signal for detection. As amplicon sizes have been getting smaller scientists have been slow to adjust the times required at each stage of the PCR accordingly.

However, demand for shorter protocols has increased, and consequently changes and innovations have been introduced to meet these demands.

The focus on shortening the time taken to perform a PCR has arisen for a number of reasons. In some laboratory settings, shorter PCR times pemiit the processing of a greater number of samples without the need to invest in additional PCR instruments, which, if a real-time capability is required, is not an insignificant cost. Also with the more frequent use of closed-tube tests, which are often monitored in real-time, the PCR step has become an increasingly large portion of the overall process and thus it becomes particularly beneficial to reduce the duration of the PCR. In addition emerging fields, such as Point-Of-Care Testing, have placed an emphasis on obtaining rapid results, again increasing the demand for shorter PCR times.

One factor that determines the speed at which a PCR can proceed is the ramp rate. The ramp rate, usually defined in °C/second, is the speed at * . * which the temperature of the sample can be altered and therefore affects the * overall time taken to perform a PCR. The time spent ramping between different temperatures can be significant, accounting for over half of the PCR in many instances. The majority of PCR instruments utilize Peltier-based heating-cooling blocks However, such blocks have relatively low ramping ** * * * rates, with 1.5-2.5 °C/second for heating and with cooling rates that are usually slower.

The demand for faster ramp rates has lead to a number of innovations, generally aimed at making heat transference more efficient. This has lead to improved ramp rates of 25-8 °Clsecond: however, these improvements impact on the instrument cost.

Alternative PCR instrument design has also lead to greatly improved ramp rates, as high as 40°Clsecond. However, these systems involve radical changes in format, away from the standard 96-well plate format which is in widespread use. They include Roche's LightCycler® instruments that utilize glass capillaries in place of the more common plastic 0.2 millilitre (mL) tube format. However, this reduces the ease by which large numbers of PCR can be set-up and also glass capillaries are easily broken which can lead to costly contamination issues. Cephied's SmartCycler® also uses dedicated plastic tubing, with a greatly improved surface to volume ratio, allowing fast ramp rates. Again, this can be a problem for those laboratories wishing to set-up large numbers of PCRs using the more common 0.2 mL tube format. The Rotorgene combines the 0.2 mL tube format with fast ramping times. However, the tubes are arranged into a circular format, making compatibility with many automated and semi-automated systems that require the use of a 96-well arrangement difficult to achieve.

Some simple steps can be taken to reduce the time of a PCR. One * . simple and commonly adopted measure to reduce PCR times is the omission *.*.** * a of the third step, to produce a two-step PCR. Thus the PCR involves repeated cycling between the denaturation step and the annealing step, as determined by the Tm of the primers. It follows that by re-designing the primers to have a *:::: Tm that is closer to the optimal working temperature for the polymerase (for example, 72°C), the temperature difference between the two steps can be significantly reduced and therefore the overall time of the PCR.

The present invention exploits these high annealing temperatures that reduce ramping times and which are closer to the optimal conditions for Taq polymerase.

BRIEF SUMMARY OF THE INVENTION

According to an aspect of the present invention, there is provided a PCR reaction mixture for quantitative detection of a specific nucleic acid sequence, comprising: a first single-stranded polynucleotide sequence comprising a primer region, a hybridisation region and a fluorophore molecule, a second single-stranded polynucleotide sequence comprising a primer region, a hybridisation region complementary to the hybridisation region of said first single-stranded polynucleotide sequence and a quencher molecule, wherein hybridisation of the hybridisation region from the first single-stranded polynucleotide sequence with the hybridisation region from the second single-stranded polynucleotide sequence brings the fluorophore molecule and quencher molecule into close proximity such that the fluorophore molecule is quenched by the quencher molecule.

According to a second aspect of the present invention, there is provided a method for quantitative detection of a specific nucleic acid sequence, comprising the steps of: i) providing a first single-stranded polynucleotide sequence comprising a primer region, a hybridisation region and a fluorophore molecule; ii) providing a second single-stranded polynucleotide sequence comprising a primer region, a hybridisation region complementary to the hybridisation region of said first polynucleoticie sequence and a quencher molecule; iii) providing a sample; iv) performing a PCR cycle; and v) measuring * . .

S

S * S

the change in light signal, either during the PCR cycle or at the end of the PCR cycle.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (a) to (d) illustrates the prior art;

Figure 2 shows the basic configuration of the oligonucleotides embodied in the present invention; Figure 3 shows an embodiment of the present invention; Figure 4 illustrates a further embodiment of the present invention; and Figure 5 shows an additional embodiment of the present invention.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

Figure 1 Many inventive and elegant methods exist that allow PCR to be monitored in real-time andfor at end-point in a closed-tube format. One method by which this can be achieved is by the introduction of a fluorescent dye e.g. SYBR®-green, into the reaction, whose properties change when intercalated into a double stranded DNA molecule, resulting in an increase of fluorescent signal. However such systems are limited to monitoring only a single PCR reaction per tube. Other methods utilize oligonucleotide probe sequences that are labelled with fluorescent dyes known as fluorophores.

The use of probe sequences permits the use of different dyes to detect :4 different sequences and so more than one reaction can be monitored in a single tube. Some methods depend on increases of fluorescent signal which are accompanied by the binding of the labelled probe to a target sequence.

However such systems have a limited increase in signal and many methods * * * . use so-called quencher molecules to create a much greater difference in **.*** * S signal between probes that are bound and those that are not bound to their intended target sequences. One such system is known as Taqman® as shown in Figure 1(a); the system comprises forward unlabelled primer 101, reverse unlabelled primer 102 and dual-labelled probe 103. Taqman® probe 103 contains a fluorescent label at one end and a quencher molecule at the other end, such that the fluorescent molecule is quenched. Taqman® probe 103 is designed to lie between the primer sites and an increase in signal is detected when the fluorophore and quencher are separated by the exonuclease activity of the DNA polymerase.

A number of methods seek to enhance the quenching of fluorophores by bring the fluorophore into close proximity to its quencher, often utilizing a stem-loop structure e.g. Amplifluor® as shown in Figure 1(b) and Scorpion1M probes (closed format) as shown in Figure 1(c). The Amplifluor® as shown in Figure 1(b) comprises forward unlabelled primer with tail 104, reverse primer 105 and dual-labelled stem-loop and primer 106 (which is identical to forward primer tail sequence 104). The Scorpion1M probe system comprises forward primer with dual-labelled stem loop probe with blocker molecule 107 and reverse primer 108. However the design and synthesis of such probes can be complex and expensive, and in the case of Scorpion1M probes, requires the introduction of a blocker molecule, which again adds to the cost of manufacture.

Another drawback of using stem-loop structures is that if a high S. *.

* annealing temperature is desired the design of such probes can become * problematic, whereby both the stem and loop need to be lengthened to ensure that the fluorescent molecules remain quenched at higher temperatures, whilst also ensuring that they adopt an unquenched format in the presence of the target sequences. Large stem-loop sequences have a greater incidence of * S unwanted secondary structures and a difficulty in maintaining the balance between the two desired states, namely quenched and unquenched.

The use of a physically separate oligonucleotide to carry the quencher molecules has been adopted by some methods, for example, displacement probes (Kaspar®) as hown in Figure 1(d). The Kaspar® system comprises forward single-labelled primer with tail sequence 109, reverse primer 110 and single-labelled oligonucleotide 111 (which is complementary to forward primer tail sequence 109). However, displacement probes such as Kaspar® can also present problems. As these quenching oligonucleotides are displaced to make way for more thermo-dynamically favourable arrangements, they remain free-floating in the reaction mixture and are still available to compete for binding with the sequence holding the fluorophore. Moreover the addition of this extra component to the reaction increases the complexity of the test and therefore the likelihood of unwanted side reactions.

Figure 2 Figure 2 shows the basic configuration of the oligoriucleotides embodied in the present invention.

The oligonucleotides embodied in the present invention overcome the problems associated with the prior art. The oligonucleotides are free from stem loop structures and the accumulation of the PCR amplicon can be monitored in real-time or at the end-point of the reaction. The oligonucleotides are 0.

* advantageously adapted for reactions with high annealing temperatures.

Furthermore, the present invention advantageously provides a means by which the fluorophore and quencher molecules are permanently separated within the reaction.

The present proposal provides a means of detecting a specific nucleic * . . S. *

S

S..... * 4

acid sequence by amplification of the nucleic acid sequence via the Polymerase Chain Reaction (PCR). Successful amplification of the sequence ia PCR results in a measureable change in light signal obtained by the stimulation and emission of light from fluorescent molecules, known as fluorophores or chromophores. Measurements can be made during the PCR process, known as real-time PCR, orat the end of the PCR process, known as end-point PCR.

The PCR reaction mixture embodied in the present invention consists of two oligonucleotides 201 and 202 which are single-stranded polynucleotide sequences, of a length typically between 30 and 50 bases. However, both shorter and longer lengths may be deployed in the present invention. Each oligonucleotide 201 and 202 sequence contains two regions; (i) a primer region and (ii) a hybridization region. The primer regions of the oligonucleotides 201 and 202 are designed such that they act as primers in the traditional sense of PCR, and allow the amplification of a target region in a specific manner, as in traditional PCR. The hybridization regions of the two oligonucleotides are complementary. One of the two oligonucleotide sequences 201 contains a fluorophore molecule and the other oligonucleotide sequence 202 contains a quencher molecule. The placement of the fluorophore and the quencher is such that they are brought into close proximity by the hybridization of the two hybridization regions contained by the oligonucleotides. This closeness of proximity causes the fluorophore molecule to be quenched by the quencher molecule. Prior to the PCR reaction this fl*** . * quenched configuration will be favoured and a background fluorescence level " 25 can be determined. 4. a.

During the denaturing step of the PCR cycle(s) the hybridized probes are separated and then they re-anneal during the annealingfextension step. I. * a

S I

However, if nucleic acid sequences are present in the reaction that are complementary to the priming regions of the oligonucleotide probes, that is, from the sample being tested, then a proportion of the oligonucleotides will anneal to these sequences. If there exists a pair of complementary regions adjacent to one another and orientated towards each other with respects to 5'- 3' extension of the priming regions, PCR will occur. These oligonucleotide sequences will become part of the PCR product or amplicon, and as such will no longer hybridise to their matching oligonueclotide. Therefore, fluorophores and quencher molecules are separated for the duration of the PCR reaction.

This separation of fluorophores is measured by the emission of light. Those molecules that do not encounter a matching sequence in the sample being tested re-anneal to their complementary oligonucleotide and are re-quenched.

Therefore, oligonucleotide sequences that do not become part of the PCR amplicon do not add to an increase in signal with respect to the background signal.

The priming region of each of the oligonucletides runs from the 3' end of each probe, for 15 to 35 bases, and typically 20 to 30 bases. In a similar manner the hybridization region of each of the oligonucleotides runs from the 5' end of each probe, for 15 to 35 bases, and typically 20 to 30 bases.

However, both shorter and longer lengths may be deployed in the present invention.

Successful amplification of a target nucleic acid sequence consequently results in the physical separation of chromophores and quencher molecules such that an increase in light can be measured when stimulation of the chromophores occurs. The present invention therefore provides a mechanism In.

by which the absence or presence of a specific nucleic acid sequence can be determined in a quantitative manner. Is.. * . . S. *

S

Figure 3 An embodiment of the present invention is shown in Figure 3. In addition to the possibility of the priming and hybridization regions of each probe being distinct from each other, as shown in Figure 2, the priming and hybridization regions of each probe may fully or partially overlap or integrate.

Figure 3a shows partial overlap of probe 301 with probe 302 whilst Figure 3b shows full integration with one primer 303 and no integration for probe 304.

Figure 3c shows full integration with both primers 305 and 306. Any region, which is not part of the priming region, is referred to as the tail region. Any region, which is not part of the priming region, or part of the hybridization region, is known as a linker region.

Figure 4 A further embodiment of the present invention is shown in Figure 4.

Oligonucleotide sequences, other than the probes, may be included in the reaction mixture, for the purposes of additional quenching. Such sequences may be composed of hybridization sequence and comprise an attached quencher molecule. In an embodiment, an excess of the quenching molecule could also meet additional quenching requirements.

* In an embodiment, the invention comprises the use of more than one fluorophore and/or quencher on each probe, as illustrated in the alternative arrangements shown in Figures 4a and 4b. In Figure 4a, a first probe comprises two fluorophores 401 whilst a second probe comprises a single quencher 402. In Figure 4b, a first probe comprises two fluorophores 403 whilst a second probe comprises two quenchers 404. In an alternative arrangement, a first probe comprises one fluorophore 405 and one quencher 406 whilst a second probe also comprises one fluorophore 405 and one quencher 406.

Figure 5 In an additional embodiment of the invention, the hybridisation sequence would be its own reverse complement. This would have several consequences. The hybridization sequence of probes would be complementary to the 3' end of the amplicon into which they become incorporated and this hybridization would be more favourable than hybridization to other complementary sequences found in the reaction, such as with other probe sequences. This would serve to ensure that probes that have become incorporated into an amplicon are prevented from being quenched, as illustrated in Figure 5 wherein complementary sequences 501 and 502 hybridise, leaving associated structures 503 and 504. Another consequence, that has been noted, of sequences which are their own reverse complement would be their tendency to form hairpin structures. * . . * * * . **** * . * .

Claims

Claims What we claim is: 1. A PCR reaction mixture for quantitative detection of a specific nucleic acid sequence, comprising: a first single-stranded polynucleotide sequence comprising a primer region, a hybridisation region and a fluorophore molecule, a second single-stranded polynucleotide sequence comprising a primer.region, a hybridisation region complementary to the hybridisation region of said first single-stranded polynucleotide sequence and a quencher molecule, wherein hybridisation of the hybridisation region from the first single-stranded polynucleotide sequence with the hybridisation region from the second single-stranded polynudeotide sequence brings the fluorophore molecule and quencher molecule into close proximity such that the fluorophore molecule is quenched by the quencher molecule.
2. The PCR reaction mixture according to claim 1, wherein said first and second single-stranded polynucleotide sequences are between 30 and 50 bases in length.
3. The PCR reaction mixture according to claim 1, wherein said * primer region of said first single-stranded polynucleotide sequence and said second single-stranded polynucleotide sequence extends from the 3' end of each single-stranded. polynucleotide sequence. * * *
4. The FCR reaction mixture according to claim 3, wherein said * . primer region extends from the 3' end of each single-stranded polynucleotide sequence for between 15 and 35 bases.
5. The PCR reaction mixture according to claim 1, wherein said hybridisation region of said first single-stranded polynucleotide sequence and said second single-stranded polynucleotide sequence extends from the 5' end of each single-stranded polyn ucleotide sequence.
6. The PCR reaction mixture according to claim 5, wherein said primer region extends from the 5' end of each single-stranded polynucleotide sequence for between 15 and 35 bases.
7. The PCR reaction mixture according to claim 1, wherein said primer region and said hybridisation region of said first single-stranded polynucleotide sequence partially overlap with said primer region and said hybridisation region of said second single-stranded polynucleotide sequence during hybridisation.
8. The PCR reaction mixture according to claim 1, wherein said primer region and said hybridisation region of said first single-stranded polynucleotide sequence fully overlap with said primer region and said * hybridisation region of said second single-stranded polynucleotide sequence * during hybridisation. *

** * .
9. The PCR reaction mixture according to claim 1, further comprising additional said second single-stranded polynucleotide sequences comprising a hybridisation region complementary to the hybridisation region of said first polynucleotide sequence and a quencher molecule.
10. The PCR reaction mixture according to claim 1, wherein said first single-stranded polynucleotide sequences comprises one or more fluorophore molecules.
11. The PCR reaction mixture according to claim 1, wherein said second single-stranded polynucleotide sequences comprises one or more quencher molecules.
12. The PCR reaction mixture according to claim 1 wherein the hybridisation region of said first single-stranded polynucleotide sequence and the hybridisation region of said second single-stranded polynucleotide sequence are reverse complement.
13. A method for quantitative detection of a specific nucleic acid sequence, comprising the steps of i) providing a first single-stranded polynucleotide sequence comprising a primer region, a hybridisation region and a fluorophore molecule; ii) providing a second single-stranded polynucleotide sequence comprising a primer region, a hybridisation region complementary to the * *: hybridisation region of said first polynucleotide sequence and a quencher * * molecule; *... ...iii) providing a sample; iv) performing a PCR cycle; and v) measuring the change in light signal, either during the PCR cycle or at the end of the PCR cycle.
14. The method according to claim 13, wherein said PCR cycle comprises a denaturing stage, an annealing stage and an extending stage.
15. The method according to claim 14, wherein said first single-stranded polynucleotide sequence and said second single-stranded polynucleotide sequence separate during said denaturing stage.
16. The method according to claim 14, wherein said first single-stranded polynucleotide sequence and said second single-stranded polynucleotide sequence re-anneal during said annealing stage and said extending stage.
17. The method according to claim 13, wherein said primer regions of said first or second single-stranded polynucleotide sequences that are complementary to nucleic acid sequences of said sample anneal to said nucleic acid sequences of said sample.
18. The method according to claim 13, wherein during said PCR reaction, said first or second single-stranded polynucleotide sequences become part of a PCR product and no longer hybridise to each other.

*
19. The method according to claim 13, wherein the separation of fluorophore and quencher molecules is quantitated by measuring the change in light signal. * * * * *
20. A PCR reaction mixture substantially as described herein and shown in the drawings.