Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA).


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Methods for Using Long ssDNA Polynucleotides as Primers in PCR Assays

FIELD OF THE INVENTION
The invention relates to nucleic acid amplification reactions and assays involving the use of long polynucleotide ssDNA as primers for monoplex and multiplex polymerase chain reactions (PCR) and, more particularly refers to methods to obtain a suitable length of a PCR product without the need to amplify longer offtarget sequences, thereby allowing the amplification of short, fragmented and degraded nucleic acids.

BACKGROUND OF THE INVENTION
Detecting and identifying variations in DNA sequences among individuals and species has provided insights into evolutionary relationships, inherited disorders, acquired disorders and others aspects of molecular genetics and medicine.

in The Fourth
International Symposium on Human Identification 1993, pp.177-187, Edwards et al. (1991Chen et al. (1993) Genomics 15(3): 621-5, Harada et al. (1994) Am. J. Hum. Genet. 55: 175-189, Comings et al. (1995), Genomics 29(2):390-6, Utah Marker Development Group (1995), Am. J. Genet. A key aspect of the methods based upon DNA length is that the products must always have a different size in order to prevent any overlapping during their detection. When using different fluorescent dyes the same concern applies to products labelled with the same dye. Since there is a limited number of fluorescent dyes that can be readily used and detected, the PCR primers in multiplex methods are designed to obtain several products of different nonoverlapping sizes for each fluorescent dye. However, this requires amplifying additional, longer off-target DNA regions in order to obtain the desired length, thereby compromising the amplification of short, fragmented and degraded DNA templates, as commonly found in DNA samples. Scientific Inc., Waltham, Massachusetts), rely on amplifying several longer offtarget sequences in order to obtain an orderly distribution of the amplicons, usually within a range from 100 to 400 base pairs.
The degradation and fragmentation of DNA samples affect the integrity and sensitivity of biomolecular assays. The patent document US 20160168644A1 teaches methods for the quantitative analysis of such nucleic acid fragmentation/degradation and its "amplificability" by determining the ratio between the amount of long and short PCR amplification products from a given sample.
The use of mini-STRs for reducing the extension of the DNA amplification, and thereby improving the PCR yield in degraded DNA samples, has been described by Whitaker JP et al. (Whitaker JP, Clayton TM, Urquhart AJ, et al. (BioTechniques (1995); 18:670-677)) and by Butler JM et al. (Butler JM, Shen Y, McCord BR. (J Forensic Sci (2003); 48). In this method the mini-STRs primers target regions adjacent to the repeats to reduce the extent of intact DNA necessary to amplify the desired repeated sequence. However, this always renders short DNA amplicons of approximately 100bp thereby limiting the number of possible mini-STRs that can be used in multiplex assays due to the overlapping effects in electrophoresis gels or CE chromatograms. Patent US 6,743,905B2 teaches methods for synthetizing and using mobilitymodifying polymers linked to a sequence-specific nucleobase polymer. By incorporating organic chains into a primer the mobility-modifying polymers can provide larger DNA amplicons without the need to actually amplify the DNA equivalent to its length, thereby reducing the extent of the amplification. Since the mobility-modifying polymer is not amplifiable, to detect the increased amplicon size the polymer is usually linked to a detection molecule, e.g. a fluorescent dye, in order to detect the DNA strand where the mobility modifier is incorporated.
This limits the length of this polymer to only short sizes to avoid the overlapping of the mobility-modified polymer primer with the labelled PCR products.  It would therefore be convenient to have a method to obtain a suitable length of a PCR product without the need to amplify longer off-target sequences, focusing on amplifying only the specific, informative target sequences.

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It is an object of the present invention to provide a use of long, single stranded DNA (ssDNA) polynucleotides as primers, instead of the usual shorter DNA oligonucleotides, yielding larger amplicons without the need to actually amplify extra DNA regions. In reducing the required template length between a given pair of primers, the performance of a PCR assay is greatly increased, particularly in degraded DNA samples, as previously shown by the use of mini-STRs.
It is another object of the present invention to provide a method to perform a polymerase chain reaction (PCR) comprising the following steps: (a) Providing a nucleic acid sample; (b) Hybridizing said nucleic acid sample to one or more pair of primers where at least one primer consists of a single stranded DNA polynucleotide having a length of 60 or more nucleotides; (c) Subjecting said nucleic acid sample to a PCR, wherein the reaction mixture medium contains at least one of said primers; and (d) Detecting the length of the amplified products.
It is still another object of the invention to provide a method to perform a polymerase chain reaction (PCR) wherein the target sequence can be amplified from any nucleic acid suitable for the PCR technique, for instance genomic DNA, mitochondrial DNA, plasmid DNA, viral DNA or RNA, synthetic DNA and messenger RNA. The preferred samples to amplify are degraded or fragmented nucleic acids encoding genetic marker sequences. The method allows to obtain amplicons of a suitable size for their detection in the presence of a degraded or fragmented target sequence. The fragmentation may have been caused by any physical or chemical effect on these target sequences.
It is even another object of the invention to provide a method to perform a polymerase chain reaction (PCR) wherein at least one primer has a length between 60 and 200 nucleotides. It may be obvious for any person skilled in the art that 6 primers longer than 200 nucleotides may also be used in the described method, these primers being accounted for in the method of invention. The long ssDNA primers may or may not be fully complementary to the target sequence, for instance while having a 3' priming, complementary sequence to the target, they may have heterologous, non-complementary sequences at the 5'-end region. One or some of the primers present in the reaction may be labelled by several means, for instance by attaching fluorescent dyes, luminescent moieties, antigens, haptens or any other signaling tags.
It is still another object of the invention to provide a method to perform a polymerase chain reaction (PCR) wherein the method may involve the hybridization of several pair of primers that amplify several different target sequences of nucleic acids at the same time, where each target sequence is a genetic marker; and where the primers and the target sequences are in the same reaction mixture. Therefore, the method can be used in different PCR formats with a different number of pair of primers as in monoplex, duplexes, triplexes and multiplexes assays.
It is another object of the invention to provide a method to detect a genetic marker in a degraded nucleic acid sample employing the polymerase chain reaction (PCR), comprising the following steps: (a) Providing a nucleic acid sample that contains one or more genetic markers; (b) Hybridizing said nucleic acid sample to one or more pair of primers where at least one primer consists of a single stranded DNA polynucleotide having a length of 60 or more nucleotides; (c) Subjecting said nucleic acid sample to a PCR, wherein the reaction mixture medium contains at least one of said primers; and (d) Detecting the length of the amplified products. It is a further object of the invention to provide a PCR kit containing one or more pair of primers, where at least one of the primers has a length of 60 nucleotides or more.
It is also an object of the invention to provide a PCR kit including a DNA polymerase enzyme or enzymes, dNTPs (deoxynucleotide triphosphate), buffers, salts, control template, size standards and all other components required to amplify and detect PCR products.

BRIEF DESCRIPTION OF THE DRAWINGS
The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, together with objects, features and advantages thereof, may be best understood by reference to the following drawings in which:         Figure 9B) shows that the peak signals decrease or disappear as the DNA is more degraded, especially for the longer amplicons, while the peak corresponding to the CSFFW200/CSFRV60J pair is still visible for the most degraded DNA.

DETAILED DESCRIPTION OF THE INVENTION
A genetic marker is understood as a gene or DNA sequence with a known location on a genome that can be used to identify a region associated to a given trait or phenotype, to inherited diseases and to the identification of individuals or species.
The term locus is sometimes used to express the same concept.
A target sequence refers to a segment of nucleic acid of interest that is selectively amplified using a sequence-specific primer.
A primer is an oligonucleotide or polynucleotide capable of hybridizing to a complimentary segment of nucleic acid to allow the initiation of the replication by a DNA polymerase enzyme.
For the purpose of the present description, the term superprimer refers to a single stranded DNA (ssDNA) polynucleotide primer longer than 60 nucleotides, more preferably between 60 and 300 nucleotides. The method demonstrated that long ssDNA primers can be readily used for PCR assays in monoplex or multiplex formats. While we preferred hot start PCR, standard PCR may also be used. Actually no special PCR conditions or primer design were needed towards that goal other than the use of highly purified, long ssDNA (superprimer) as primer reagent. Moreover, we also showed that these primers can be applied in the amplification of degraded DNA templates with the same beneficial effect of the small mini-STRs, while at the same time producing larger DNA amplicons of flexible length that are more suitable for size discrimination assays.
Primers totally complementary to the template sequence or complementary only at their 3' priming region were both successfully amplified. The use of nonhomologous sequences may be important for providing flexibility in designing multiplex reactions with several primers. Furthermore, primers bearing long 5', non-homologous sequences might be considered for performing mutagenesis and also for annealing, molecular binding and labeling strategies.
The invention method is suitable for PCR assays and for detecting variations that involve length polymorphisms. It may be applied to the current methods in use that rely on different amplification lengths to distinguish variations in nucleic acid sequences. It may also be useful in the design of multiplex kits for the identification of individuals and species and for medical diagnostics.
The invention method increases the sensitivity of PCR assays by reducing the required length of available intact DNA template needed for amplification while at the same time obtaining larger DNA amplicons of flexible length suitable for size discrimination in analysis by CE and other related methods. For that aim, the addition of long ssDNA fragments as primers (superprimers) allows the enzymatic polymerization of specific, small DNA target regions while at the same time yielding product sizes suitable for their convenient detection.

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The present invention increases the sensitivity of PCR assays by reducing the required length for intact DNA template while at the same time obtaining larger DNA amplicons suitable for size discrimination.
The method of the invention is useful to obtain a suitable length of a PCR product without the need to amplify off-target sequences towards that aim, thereby increasing the sensitivity of the assay for short, fragmented or degraded DNA  Long ssDNA primers produced neat PCR products in a similar fashion to shorter oligonucleotide primers, without any artifacts or spurious bands. Figure  PowerPlex®16HS, even though the latter amplicon is 25 bases shorter (Fig. 2B).
All profiles agreed with the ones described in the literature for these cell types Other commonly used STR loci as DYS391 and Penta E can be successfully amplified and combined in multiplex formats using long ssDNA primers (superprimers). Figure 4A shows the amplification products for locus CSF1PO, Penta E and DYS391 on 2800M DNA using the following sets of primers pairs: CSFFW120/CSFRV60J, PEFW120/PERV60F and DY391F2/DSYRV120 (Fig.   4B). These primer pairs were in turn successfully combined to produce all possible duplex combinations and a triplex. Figure 5 shows the CE profile of the triplex assay with the expected peak profiles for DYS391 and Penta E (FAM channel) and CSF1PO (JOE channel).
Long ssDNA primers (superprimers) with partial, non-complementary sequences may also be successfully utilized. Figure 6 shows Long ssDNA primers (superprimers) are compatible with more complex PCR multiplex assays. In order to know if the long ssDNA fragments (superprimers) might somehow interfere in assays involving several primers, we added primer pairs DYS391F2/DYSRV120 and DYS391F2/DYSRV200 into a commercial kit already containing 42 primers (PowerPlex® 21, Promega, with 42 primers for amplifying 21 loci (Fig. 7A). Figure 7B shows that the 2800M DNA profile for all 21 markers is not altered by the addition of the long primers, while a peak corresponding to DYS391 is identified in the FAM channel at the expected size for both pairs. Superprimers added to an existing kit provide the expected results without interfering in the multiplex detection of several loci. The method of the invention may be utilized in multiplex PCR assays.
Long ssDNA primers (superprimers) may be useful for amplifying degraded DNA. To demonstrate the advantage of using long primers on fragmented or degraded DNA samples, genomic DNA was subjected to a controlled degradation by heating at 95ºC at increasing periods of time. The resulting degraded DNA was qPCR amplified using either standard PowerPlex®16HS primers or the longer ssDNA pair of primers (superprimers) CSFFW200 (200nt)/CSFRV60J (60nt) ( Figure 2B). Figure 8 shows that for more degraded DNA the Ct in the reaction with long ssDNA primers becomes lower than in the one with the standard PowerPlex®16HS primers, illustrating that they are more suitable for amplifying degraded DNA.
Long ssDNA primers are helpful to detect genetic markers on complex multiplex assays involving dozens of primers when the DNA sample is degraded. For instance, superprimers can be used in the current kits containing the CODIS (Combined DNA Index System) markers of the FBI (Federal Bureau of Investigation) database in order to amplify loci that otherwise might remain undetected, especially those ones that have large amplicon sizes. Figure 9A shows the qPCR profiles of 2800M DNA heated at 95ºC for increasing periods of time and then amplified using primers CSFFW200 (200nt) and JOE-labelled CSFRV60J (60nt). In spite that the DNA gets more degraded by longer heating incubations, the primer pair CSFFW200/CSFRV60J yields a PCR product in all time points. Degraded DNA was used -along an NTC (no template control) -to demonstrate the advantage of using superprimers in a current CODIS (Combined DNA Index System, FBI) commercial kit. To that aim the CSFFW200/CSFRV60J pair was added to the PowerPlex® Fusion 6C reaction mix, which already contains 54 primers that amplify 27 loci. The peak signals decrease as the DNA is more degraded, especially for the longer amplicons ( Figure 9B). However, degraded samples amplified with long ssDNA primers CSFFW200/CSFRV60J still show useful genotyping peaks for the CSF1PO marker. This demonstrates the advantage of the superprimers when performing complex CODIS multiplex assays in the presence of degraded DNA samples.
As the DNA is degraded the longer primers are more efficient in amplifying the resulting fragmented DNA templates due to the shorter amplification span between the 3' end of the forward and reverse primers. This is the same effect as The electrophoresis gel shows that long ssDNA primers produced neat PCR products in similar fashion to shorter oligonucleotide primers, without any artifacts or spurious bands (Figure 2A).
Moreover, they showed the characteristic heterozygous unbalance present in cell lines K562 (dominant 10 in 9, 10) and 9948 (dominant 10 in 10, 11). There is also a small peak in cell line 9948 (Fig. 3B) that may represent the allele 12 triplet of CSF1PO which has a relative intensity less than 10 % of the dominant allele 10.

EXAMPLE 3
Amplification of STR loci in a Multiplex Format. Figure 4A shows the amplification products for locus CSF1PO, Penta E and DYS391 on 2800M DNA using the following sets of primers pairs: CSFFW120 / CSFRV60J, PEFW120 / PERV60F and DY391F2 / DSYRV120 (Fig. 4B). These primer pairs were in turn successfully combined to produce all possible duplex combinations and a triplex.   EXAMPLE 5

Addition of Long ssDNA Primers in Complex Multiplex PCR Assays
In order to know if the long ssDNA fragments might somehow interfere in assays involving several primers, we added primer pairs DYS391F2/ DYSRV120   2. The method according to claim 1 wherein the nucleic acid is genomic DNA.
3. The method according to claim 2 wherein the sequence of genomic DNA is a genetic marker selected from the group consisting of genes, identification markers and inherited disease markers.
4. The method according to claim 2 wherein the nucleic acid is degraded or fragmented.
5. The method according to claim 1 wherein at least one primer has a length between 60 and 300 nucleotides.
6. The method according to claim 1 wherein the sequence of at least one of the primers is fully complementary to the target sequence. 27 7. The method according to claim 1 wherein at least one of the primers has nonhomologous sequences at the 5' end, having its 3´-priming region complementary to the target sequence.
8. The method according to claim 1 wherein the target sequence is a STR genetic marker selected from the group consisting of CSF1PO, DYS391 and Penta E and a combination thereof.
9. The method according to claim 1 wherein said method comprises the hybridization of more than one pair of primers in order to simultaneously amplify more than one target sequence, where each target sequence is a different genetic marker, being all the primers and target sequences in the same reaction mixture.
10. The method according to claim 1 wherein the PCR assay is selected from a group consisting of monoplex, multiplex, end-point, real-time, hot start and nonhot start.
11. The method according to claim 1 wherein at least one primer is labelled by one of the group consisting of a fluorescent dye, luminescent moiety, enzyme, hapten and antigen.
12. The method according to claim 1 wherein the nucleic acid is selected from the group consisting of mitochondrial DNA, plasmid DNA, viral DNA or RNA, synthetic DNA and messenger RNA.
13. A method to detect in a degraded nucleic acid sample one or more genetic markers by performing a polymerase chain reaction (PCR) comprising the following steps: a. Providing a nucleic acid sample; 28 b. Hybridizing said degraded nucleic acid sample to one or more pair of primers where at least one primer consists of a single stranded DNA polynucleotide having a length of 60 or more nucleotides; c. Subjecting said nucleic acid sample to a PCR, wherein the reaction mixture medium contains at least one of said primers; and d. Detecting the amplified products.
14. The method according to claim 13 wherein the target sequence is a STR genetic marker selected from the group consisting of CSF1PO, DYS391, Penta E and a combination thereof.
15. The method according to claim 13 wherein at least one primer has a length between 60 and 300 nucleotides.
16. The method according to claim 13 wherein the sequence of at least one of the primers is fully complementary to the target sequence.
17. The method according to claim 1 wherein at least one of the primers has nonhomologous sequences at the 5' end, having its 3´-priming region complementary to the target sequence.
18. The method according to claim 13 wherein at least one primer is labelled by one of the group consisting of fluorescent dye, luminescent moiety, enzyme, hapten and antigen.
19. The method according to claim 13 wherein the PCR assay is selected from a group consisting of monoplex, multiplex, end-point, real-time, hot start and nonhot start. 29 20. A kit comprising of at least one pair of primers, wherein at least one primer has a length of 60 nucleotides or more.

21.
A kit according to claim 20 wherein said kit comprises more than one pair of primers in order to simultaneously amplify more than one target sequence, where each target sequence is a different genetic marker, being all the primers and target sequences in the same reaction mixture.

ABSTRACT
Method to perform a PCR assay that comprises the following steps: a. Obtaining a nucleic acid sample; b. Hybridizing that nucleic acid sample to one or more pair of primers where at least one primer consists of a single stranded DNA polynucleotide having a length of 60 or more nucleotides; c Subjecting said nucleic acid sample to a PCR, wherein the reaction mixture medium contains at least one of said primers; and d. Detecting the length of the amplified products.
The amplified nucleic acid may contain any sequence or multiple sequences of STRs (short tandem repeats), genes or any coding region having a defined location on a genome. The preferred nucleic acid samples to be amplified are degraded or fragmented and contain one or more genetic markers.