WO2015063154A1

WO2015063154A1 - Blocking primers in multiplex pcr based assays

Info

Publication number: WO2015063154A1
Application number: PCT/EP2014/073222
Authority: WO
Inventors: Jurgen Peter Lode DEL FAVERO
Original assignee: Multiplicom N.V.
Priority date: 2013-10-31
Filing date: 2014-10-29
Publication date: 2015-05-07

Abstract

The present invention relates to methods for performing multiplex polymerase chain (PCR) reactions and to nucleic acid amplification mixtures suitable to be used in the present methods. The present invention further relates to the use of oligonucleotides or blocking primers in a multiplex polymerase chain reaction for preventing or reducing primer-dimer formation, for blocking of specific amplification fragments or for stabilization of specific amplification fragments in said multiplex polymerase chain reaction.

Description

BLOCKING PRIMERS IN MULTIPLEX PCR BASED ASSAYS Description

The present invention relates to methods for performing multiplex polymerase chain (PCR) reactions and to nucleic acid amplification mixtures suitable to be used in the present methods. The present invention further relates to the use of oligonucleotides in a multiplex polymerase chain reaction for preventing, or reducing, primer-dimer formation, for blocking of specific amplification fragments or for stabilization of specific amplification fragments in a multiplex polymerase chain reaction.

Multiplex polymerase chain reaction (mPCR) is a variant of PCR in which two or more target sequences can be amplified by including more than one pair of nucleic acid amplification, or PCR, primers in a single nucleic acid amplification reaction.

Multiplex PCR has the potential to produce

considerable savings of time and effort in the laboratory. Since it was first described in 1988, this technique has been successfully applied in many areas of DNA testing, including gene deletion analysis, mutation and polymorphism analysis , quantitative analysis , and reverse-transcription (RT ) -PCR . In the field of infectious diseases, multiplex PCR has been shown to be a valuable tool for identification of viruses, bacteria, and parasites.

The optimization of mPCR poses several

difficulties, including poor sensitivity and specificity, and/or preferential amplification of certain specific targets . The presence of more than one primer pair in the multiplex PCR increases the chance of obtaining spurious amplification products , for example, due to the formation of primer-dimers .

An important concept in PCR is an optimal primer- to-template ratio. If the ratio is too high, primer-dimers are formed, as also occurs in conditions of very dilute template or excess primer. Primers must usually be in molar excess with respect to a template. Another factor

determining the primer-to-template ratio is the amount and complexity of the template to be amplified. If the primer- to-template ratio is too low, primer-dimers may be formed more efficiently than the desired target.

The optimization of multiplex PCR should aim to minimize nonspecific interactions. Special attention to primer design parameters , such as homology of primers with their target nucleic acid sequences, length, GC content, and concentration, have to be considered. Hot start PCR can eliminate nonspecific reactions caused by primer annealing at low temperature.

Ideally, all the primer pairs in a multiplex PCR should enable similar amplification efficiencies for their respective targets. Theoretically, this may be achieved through the utilization of primers with nearly identical optimum annealing temperatures displaying no significant homology either internally or to one another .

Preferential amplification of one target sequence over another is a known phenomenon in mPCR . Two major classes of processes that induce this bias have been

identified : PCR drift and PCR selection . PCR drift is a bias assumed to be due to fluctuation in the interactions of PCR reagents , particularly in the early cycles , especially in the presence of very low template concentration . PCR

selection, on the other hand, is defined as a mechanism that favors the amplification of certain templates due to the properties of the target , the target ' s flanking sequences , or the entire target genome. These properties include interregional differences in GC content, leading to

preferential accessibility of targets within genomes due to secondary structures and the gene copy number within a genome .

In summary, the most common problems of establishing multiplex PGR based tests are: spurious amplification products including primer dimer products , uneven or no amplification of some target sequences, and poor reproducibility.

It is an object of the present invention, amongst other objects, to obviate at least part, if not all, of the above problems associated with multiplex polymerase chain reactions .

The above object, amongst other objects, is met by the present invention as outlined in the appended claims.

Specifically, the above object, amongst other objects, is met by the present invention, according to a first aspect, by methods for performing a multiplex

polymerase chain reactions comprising the steps of:

a) preparing a multiplex polymerase chain

reaction mixture comprising polymerase chain reaction primers; and

b) performing a multiplex polymerase chain

reaction on the multiplex polymerase chain reaction mixture;

wherein the above mixture comprises oligonucleotides having the complementary sequence of at least one of said

polymerase chain reaction primers at, or near, the 3' side of the PCR primers and these oligonucleotides further have 3' blocking groups preventing nucleic acid extension of the oligonucleotides . The present inventors surprisingly discovered that including one or more complementary oligonucleotides, also designated herein as blocking primers, in a nucleic acid amplification mixture for multiplex PCR, or mPCR reaction, provides a clear and reproducible effect on the targeted PCR primers resulting in stabilization, reduction or blocking of the targeted primer (s) and/or prevention of primer-dimer formation .

The present inclusion of complementary

oligonucleotides in a multiplex amplification reaction mixture is especially beneficial in case of complex

multiplex polymerase chain reaction involving selective and simultaneous amplification of 5 or more amplicons, or targets, such as more than 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100.

Within the context of the present invention, it is contemplated that the present inclusion of complementary oligonucleotides, or blocking primers, could provide a reliable and effective nucleic acid amplification of 100 or more amplicons, or targets, such as more than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or even higher in a single nucleic acid amplification reaction or multiplex PCR (mPCR) amplification.

Within the present context, a blocking primer is defined as an oligonucleotide that has the complementary sequence (5' to 3') starting at, or close to, the 3' part of a PCR primer such as 0, 1, 2, 3, 4, or 5 bp from the 3' end of a PCR primer. Furthermore, the present blocking primers contain a blocking group added to its 3' end for preventing elongation, or extension, of the blocking primer during the amplification reaction. As a result of the blocking group, a blocking primer can anneal to primers and/or template sequences but can not be extended during PCR cycling. According to the present invention, any multiplex polymerase chain reaction mixture can be used. In general, such mixture comprises, besides the present blocking

primers, at least two or more PCR primer pairs for

amplification of two or more target sequences, or amplicons, a polymerase, suitable nucleotides, a buffer, optional additives and a template to be amplified. Generally, also divalent cations such as Mg²⁺ and monovalent cations such as potassium ions will be present in the reaction mixture.

For performing the present multiplex polymerase chain reaction use can be made of any suitable, and known, apparatus such as a thermocycler .

The present blocking primers can be used in any suitable ratio as compared to the specific PCR primer to be blocked such as ratios of 0.01:1 to 100:1. Contemplated ratios within the context of the present invention include 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1 or 1.9:1 but also 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1 or 0.09:1. Additionally contemplated ratios include 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 or 90:1.

The number of blocking primers to be included in the present multiplex amplification reaction mixture is dependent on the PCR primers used, the reaction conditions such as hot start or annealing temperature, the amount of template, the number of amplicons to be amplified etc. A skilled person is readily able to determine which PCR primer, or PCR primers, are biasing the multiplex

amplification and, accordingly, require the addition of the present blocking primers to the reaction mixture.

Accordingly, the number of blocking primers to be added to the present reaction mixture depends on the PCR primer pairs used. For example, if n PCR primer pairs are used. The n can be any integer between 2n and 1, wherein n≥2, i.e. blocking of all primers in the reaction (2n) versus blocking a single primer (1) in a multiplex PCR reaction.

According to an embodiment of this first aspect of the present invention, the length (in base pairs or bp) of the present blocking primers is in the range of 20% to 100% of the length (in base pairs or bp) of the PCR primers used to amplify the specific target sequences in the template. Suitable percentages include 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

As an example, if the length of a PCR primer is 50 base pairs, or bp, a suitable length of the blocking primer will be in the range of 10 (20%) to 50 (100%) bp.

According to a preferred embodiment of this first aspect of the present invention the length of the present blocking primers is in the range of 50% to 100% of the length of the specific PCR primers or, according to an especially preferred embodiment, the length of the present blocking primers is approximately 50%, 70% or 100% of the length of specific PCR primers.

According to another preferred embodiment of this first aspect of the present invention, the present multiplex polymerase chain reaction mixture comprises at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 specific polymerase chain reaction primer pairs such as at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or even higher.

According to a second aspect, the present invention relates to the use of the present blocking primers in multiplex polymerase chain reaction for preventing or reducing primer-dimer formation in the multiplex polymerase chain reaction. According to embodiment of this second aspect, the blocking primers have a length being in the range of 20% to 100% of the length of the polymerase chain reaction primers such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, preferably the oligonucleotides have a length of approximately 70% or 50% of the length of the polymerase chain reaction primers.

According to a third aspect, the present invention relates to the use of the present blocking primers in a multiplex polymerase chain reaction for blocking of specific amplification fragments in the multiplex polymerase chain reaction .

According to embodiment of this third aspect, the blocking primers have a length being in the range of in the range of 20% to 100% of the length of the polymerase chain reaction primers such as more than 30%, 40%, 50%, 60%, 70%, 80%, 90% of the length of the polymerase chain reaction primers, preferably the oligonucleotides have substantially the same length of the polymerase chain reaction primers.

According to a fourth aspect, the present invention relates to the use of the present blocking primers in a multiplex polymerase chain reaction for stabilization of specific amplification fragments in the multiplex

polymerase chain reaction.

According to embodiment of this fourth aspect, the blocking primers have a length being in the range of in the range of 20% to 100% of the length of the polymerase chain reaction primers such as more than 30%, 40%, 50%, 60%, 70%, 80%, 90% of the length of the polymerase chain reaction primers, preferably the blocking primers have substantially the same length of the PCR primers or approximately 50% to 70% of the length of the polymerase chain reaction primers or a length being in the range of 70% to 100% of the length of the polymerase chain reaction primers. According to a fifth aspect, the present invention relates to nucleic acid amplification mixtures suitable for performing a multiplex polymerase chain reaction wherein the mixture comprises oligonucleotides having the complement sequence of, or close to, the 3' part of at least one of the polymerase chain reaction primers in the mixture and the oligonucleotides further having 3' blocking groups.

In general, the present amplification mixtures comprise, besides the present blocking primers, at least two or more PCR primer pairs, such as more than 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900. 1000, 1500, 2000 or higher for amplification of two or more target sequences, or amplicons, a polymerase, suitable nucleotides and a buffer. Generally, also divalent cations such as Mg²⁺ and monovalent cations such as potassium ions will be present in the reaction mixture.

According to an embodiment of this fifth aspect of the present invention, in the present nucleic acid

amplification mixture the length of the present blocking primers is in the range of 20% to 100% of the length of the polymerase chain reaction primers such as more than 30%, 40%, 50%, 60%, 70%, 80%, 90% of this length, preferably the length of the present blocking primers is in the range of 50% to 70% or 70% to 100% of the length of the polymerase chain reaction primers, more preferably approximately 50%, 70% or 100%.

According to an especially preferred embodiment of this fifth aspect of the present invention, the present nucleic acid amplification mixture comprises at least 3 polymerase chain reaction primer pairs each capable of amplifying a specific template target such as at least 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 50, 100, 150, 200, 300, 4000, 500, 600, 700, 800, 900, 1000, 1500, 2000 or higher. The present nucleic acid amplification mixture, according to an especially preferred embodiment, is used for the non-invasive detection of foetal chromosomal

abnormalities in a sample derived from a pregnant female, preferably chromosomal aneuploidy.

In the above use, the sample is preferably derived from, or finds its origin in, blood from a pregnant female.

According to a most preferred embodiment of all the above aspects of the present invention, the present sample comprises cell-free DNA or free-floating DNA of a foetus to be amplified.

The present invention will be further detailed in the following examples of preferred embodiments of the present invention. In the examples, reference is made to figures wherein:

Figure 1 : shows the result of a multiplex PGR using blocking primers with a length of approximately 70% (excluding adaptor sequences) the length of the PCR primer .

Figure 2 : shows the result of a multiplex PCR using different ratios of blocking primers and PCR primers .

Examples

Example 1 : Prevention or reduction of primer-dimer

formation observed in mPCR reactions

Prevention or reduction of primer-dimer formation observed in mPCR reactions is realized by adding blocking primers designed to bind the PCR primer (s) that lead to primer-dimer formation in a tested ratio PCR primer v.s. blocking primer of respectively: 1/1; 1/0.5 or 1/0.1 (see Figure 1) .

Specifically, in Figure 1A: no blocking primer is added and shows a primer-dimer problem (arrow) in a

multiplex PCR reaction. The peaks in the grey bars represent the expected amplicons, which show very low amplification in the absence of blocking primers of 14 bp. In this experiment blocking primers were designed and added for 3 primer pairs that resulted in the formation of the observed primer-dimer. Figure IB: 1/1 ratio PCR primer and blocking primer; Figure 1C : 1/0.5 ratio PCR primer and blocking primer; Figure ID: 1/0.1 ratio PCR primer and blocking primer.

Adding blocking primers of 14 bp in length to the mPCR clearly shows that the primer-dimer peak is no longer present and hence the amplification of the mPCR amplicons in Figures B, C and D is more efficient compared to A were no blocking primer was added. Furthermore, no effect of the blocking primer is observed on the ratio of the amplicons within the mPCR.

In conclusion, in this example, it was observed that for prevention or reduction of primer-dimer formation the optimal length of the blocking primer is ~ 70% of the PCR primer (e.g. PCR primer 20 bp and blocking primers is 14 bp) .

In some cases it was observed that a mismatched 3 ' base of the blocking primer results in more efficient primer-dimer reduction (data not shown) . In this case the blocking primer is 15 bp long: 14 bp complementary to the PCR primer and the 3' base, on which the blocking group is covalently bound, is mismatched with the template PCR primer . Example 2: Complete blocking of specific amplicons in mPCR

Complete blocking of specific amplicons in mPCR was realized by adding blocking primers, designed to bind the PCR primer (s) in a tested ratio PCR primer v.s. blocking primer of respectively: 1/1; 1/5. Figure 2 shows the results of adding blocking primers in a ratio of 1/1. Data from the ratio 1/5 are not shown since they are identical to the 1/1 ratio.

Specifically, Figure 2 shows the effect of full blocking primer on amplification of blocked amplicons. In Figure 2A no blocking primer was added (reference pattern) . The peaks in the grey bars represent the expected amplicons. In Figures 2B-D : 1/1 ratio of PCR primers and blocking primers; B: full blocking primers added for amplicons 1, 2, 3 and 4; C: full blocking primers added for amplicons 1, 2, 3, 4, 5 and 6; D: full blocking primers added for all amplicons .

In conclusion, Figure 2 shows that adding full blocking primers to the mPCR clearly provides that targeted amplicons are not amplified. Furthermore, no effect of the blocking is observed on the relative ratio of the remaining amplicons within the mPCR.

The optimal (tested) length of the blocking primer is equal to the length of the PCR primer (i.e. full blocking primer )

Example 3 Stabilisation of specific amplicons in a mPCR

It is observed in mPCR that occasional primer sets amplify more efficiently compared to other amplicons in a mPCR. Such primer sets are referred to as 'reactive

amplicons'. The problem with reactive amplicons is that lowering the primer concentration results in failing

amplification of the corresponding amplicons, resulting in a difficult to optimize mPCR.

Adding blocking primers should allow the transformation of reactive amplicons into amplicons behaving as standard amplicons.

Claims

1. Method for performing a multiplex polymerase chain reaction comprising :

a) preparing a multiplex polymerase chain

reaction mixture comprising polymerase chain reaction primers

b) performing a multiplex polymerase chain

reaction on said mixture;

wherein said mixture comprises oligonucleotides having the complementary sequence of the 3 ' end of at least one of said polymerase chain reaction primers and said oligonucleotides further having 3' blocking groups.

2. Method according to claim 1, wherein the length of said oligonucleotides is in the range of 20% to 100% of the length of said polymerase chain reaction primers.

3. Method according to claim 1 or claim 2, wherein, the length of said oligonucleotides is in the range of 50% to 100% of the length of said polymerase chain reaction primers.

4. Method according to any one of claims 1 to 3, wherein the length of said oligonucleotides is approximately 70% or approximately 100% of the length of said polymerase chain reaction primers.

5. Method according to any one of the claim 1 to 4, wherein said multiplex polymerase chain reaction mixture comprising at least 3 polymerase chain reaction primer pairs .

6. Use of oligonucleotides having the complementary sequence of the 3 ' end of at least one of the polymerase chain reaction primers used in a multiplex polymerase chain reaction and said oligonucleotides further having 3' blocking groups for preventing or reducing primer- dimer formation in said multiplex polymerase chain reaction.

7. Use according to claim 6, wherein said oligonucleotides have a length being in the range of 20% to 100% of the length of said polymerase chain reaction

primers .

8. Use according to claim 6 or claim 7, wherein said oligonucleotides have a length of approximately 70% of the length of said polymerase chain reaction primers.

9. Use of oligonucleotides having the

complementary sequence of at least one of the polymerase chain reaction primers used in a multiplex polymerase chain reaction and said oligonucleotides further having 3 ' blocking groups for blocking of specific amplification fragments in said multiplex polymerase chain reaction.

10. Use according to claim 9, wherein said

oligonucleotides have a length being in the range of 20% to 100% of the length of said polymerase chain reaction

primers .

11. Use according to claim 9 or claim 10, wherein said oligonucleotides have substantially the same length of said polymerase chain reaction primers.

12. Use of oligonucleotides having the complementary sequence of the 3 ' end of at least one of the PCR primers used in a multiplex polymerase chain reaction and said oligonucleotides further having 3' blocking groups for stabilization of specific amplification fragments in said multiplex polymerase chain reaction.

13. Use according to claim 12, wherein said oligonucleotides have a length being in the range of 20% to 100% of the length of said polymerase chain reaction

primers .

14. Use according to claim 12 or claim 13, wherein said oligonucleotides have substantially the same length of said PCR primers or approximately 70% of the length of said polymerase chain reaction primers or a length being in the range of 70% to 100% of the length of said polymerase chain reaction primers.

15. Nucleic acid amplification mixture suitable for performing a multiplex polymerase chain reaction said mixture comprises oligonucleotides having the complementary sequence of the or close to the 3 ' end of at least one of the polymerase chain reaction primers in said mixture and said oligonucleotides further having 3' blocking groups.

16. Nucleic acid amplification mixture according to claim 15, wherein the length of said oligonucleotides is in the range of 20% to 100% of the length of said polymerase chain reaction primers.

17. Nucleic acid amplification mixture claim 15 or claim 16, wherein, the length of said oligonucleotides is in the range of 70% to 100% of the length of said polymerase chain reaction primers, preferably approximately 70% or 100%.

18. Nucleic acid amplification mixture comprising at least 3 to 1000 polymerase chain reaction primer pairs.

19. Use of a nucleic acid amplification mixture according to any one of the claims 15 to 18 for the non^¬ invasive detection of foetal chromosomal abnormalities in a sample derived from a pregnant female.

20. Use according to claim 19, wherein said foetal chromosomal abnormalities are chromosomal aneuploidy.

21. Use according to claim 19 or claim 20, wherein said sample is derived from blood.

22. Use according to any one of the claims 19 to 21, wherein said sample comprises cell-free DNA or free- floating DNA of a foetus.