Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA

Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67 bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.     

应用改进的通用荧光PCR引物进行多重STR分型

通过设计通用荧光PCR引物并结合DNA测序系统建立了小鼠的多重STR分型方案.实验针对小鼠基因组设计了两对不同的通用引物序列,标记了FAM荧光的通用序列和"加尾"的位点特异性引物共同用于小鼠的多重PCR的STR基因分型.本研究优化了通用引物和特异性引物间的比例,优化了多重STR-PCR的反应条件,并最终利用该技术方案实现了五重STR分型.实验验证了该方案在多重STR分型中的可行性.与传统的荧光检测PCR产物方案相比,应用通用方案完成多重PCR反应大大节省了实验时间与经费.     

Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing.

We describe a simple and cost-effective method for the synthesis of an internal fluorescently labeled DNA standard for fragment sizing using an automated DNA sequencer. A set of primer pairs labeled with ROX was developed to amplify 12 DNA fragments, 58-417 bp, derived from a conserved region of plant chloroplast DNA. These amplified fragments were mixed together, constituting a fluorescent internal DNA size marker. The precision of the size standard was evaluated by estimating the size of 20 alleles that were amplified at four dinucleotide microsatellite loci with the synthesized size standard and the commercial internal sizing standard, GeneScan Rox500. A number of intra-gel and inter-gel comparisons were run, and an analysis of variance was carried out. No significant difference was observed between the size estimates obtained with the synthesized DNA standard and the commercial standard. This facile and general PCR-based method for the synthesis of internal standards allows for significant savings in the implementation of large genotyping experiments using microsatellite or AFLP markers.     

Highly accurate SNP genotyping from historical and low‐quality samples

Historical and other poor‐quality samples are often necessary for population genetics, conservation, and forensics studies. Although there is a long history of using mtDNA from such samples, obtaining and genotyping nuclear loci have been considered difficult and error‐prone at best, and impossible at worst. The primary issues are the amount of nuclear DNA available for genotyping, and the degradation of the DNA into small fragments. Single nucleotide polymorphisms offer potential advantages for assaying nuclear variation in historical and poor‐quality samples, because the amplified fragments can be very small, varying little or not at all in size between alleles, and can be amplified efficiently by polymerase chain reaction (PCR). We present a method for highly multiplexed PCR of SNP loci, followed by dual‐fluorescence genotyping that is very effective for genotyping poor‐quality samples, and can potentially use very little template DNA, regardless of the number of loci to be genotyped. We genotyped 19 SNP loci from DNA extracted from modern and historical bowhead whale tissue, bone and baleen samples. The PCR failure rate was < 1.5%, and the genotyping error rate was 0.1% when DNA samples contained > 10 copies/µL of a 51‐bp nuclear sequence. Among samples with ≤ 10 copies/µL DNA, samples could still be genotyped confidently with appropriate levels of replication from independent multiplex PCRs.     

Multiplex PCR-based Alu insertion polymorphisms genotyping for identifying individuals of Japanese ethnicity

Discrimination of Alu insertions is a useful tool for geographic ancestry analysis, and is usually performed by Alu element amplification and agarose gel electrophoresis. Here, we have developed a new fluorescence-based method for multiple Alu genotyping in forensic identification. Allele frequencies were determined in 70 Japanese individuals, and we selected 30 polymorphic Alu insertions. Three primers were designed for each Alu locus to discriminate alleles using the 3-6 bp differences in amplicon sizes. Furthermore, we classified the amplification primers for the 30 loci into three different sets, and PCR using each set of primers provided 10 loci fragments ranging from 50 to 137 bp. Based on population data, the probability of incorrectly assigning a match was 3.7×10(-13). Three independent amplifications and subsequent capillary electrophoresis enabled the sensitive genotyping of small amounts of DNA, indicating that this method is suitable for identifying individuals of Japanese ethnicity.     

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.     

Two highly validated SSR multiplexes (8‐plex) for Euphrates' poplar,Populus euphratica (Salicaceae)

Multiplex PCR amplification of microsatellites has significantly increased the throughput and decreased the costs of genotyping. We have developed two highly polymorphic microsatellite multiplexes for Populus euphratica, the only tree species found in desert regions of Western China and adjacent Central Asian countries. The first of these multiplex kits comprises an eight‐Plex of genomic SSRs (gSSRs) obtained from published databases. The second comprises an eight‐plex of newly designed EST‐SSRs (eSSRs) based on expressed sequence tags for P. euphratica. Both kits were tested on a sample of 170 individuals from four populations. The gSSRs exhibited slightly more polymorphism than the eSSRs. The new multiplex protocols yielded consistent results in the hands of multiple researchers, demonstrating their robustness. The 16 loci used in the kits exhibited a high transferability rate (82.0%) in eight other poplar species belonging to five different sections of the genus. Both kits should therefore be useful for further investigations of population genetics in P. euphratica and related species. Our results indicate that it is essential to follow recently established recommendations when developing microsatellite markers, including verifying the amplification efficiency, detecting null alleles and carefully measuring error rates.     

An innovative SNP genotyping method adapting to multiple platforms and throughputs

Key message

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility.

Abstract

Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3′ regions to significantly increase the amplification specificity of the two alleles and tailed at 5′ ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion–deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

     

Poly(A) tailing of ancient DNA: a method for reproducible microsatellite genotyping

Microsatellites could be of great potential use in the analysis of ancient remains, but so far such analyses have failed to be reproducible mainly because of the high degree of ancient DNA (aDNA) degradation. During PCR, annealing of the primers to the complementary sequences of microsatellites occurs together with cross-annealing of partially degraded repeated sequences. This could create chimeric alleles that do not correspond to the authentic ones. Here we report a simple method for processing aDNA fragments prior to PCR that greatly reduces the production of chimeric alleles. This approach eliminates aDNA molecules broken within the repeats as targets for Taq polymerase by adding poly(A) tails at the 3(') ends of the DNA fragments, which disrupts the homology in the region and thus prevents annealing out of register. We have analyzed one dinucleotide- (D6S337) and two trinucleotide-containing loci (IT15 and SCA1) using poly(A)-tailed and the same untreated aDNA as template. aDNAs were isolated from 28 human remains, 600 and 7000 years of age. In repeated experiments with untreated aDNAs we obtained three to five times more alleles compared to poly(A)-tailed aDNAs. According to our results, modification of aDNA by poly(A) tailing is an efficient pretreatment for accurate genotyping.     

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.     

Multiplexed microsatellite markers for genetic studies of beech

European beech (Fagus sylvatica L.) is one of the economically most important broadleaved tree species in Europe and has become a model for studying climate change effects on forests. Multiplex PCR of microsatellites is a fast and cost-effective technique allowing high-throughput genotyping. Here we present the procedure used to develop two multiplex kits (8-plexes) for European beech. We paid particular attention to quality control throughout all steps of the multiplex kits development (null allele detection, error rate measurements, linkage disequilibrium). Preliminary assays suggest that the 16 amplified loci are largely devoid of null alleles and allow rapid and cost-effective genotyping of beech with low error rates. The two kits, which differ in their levels of polymorphism, most likely due to marker origin, were also informative in seven other beech species tested.     

Multiplex SNP-SCALE: a cost-effective medium-throughput single nucleotide polymorphism genotyping method

We describe a convenient, cost-effective and flexible medium-throughput single nucleotide polymorphism (SNP) genotyping method, Multiplex SNP-SCALE, which enables the simultaneous amplification by polymerase chain reaction (PCR) of up to 25 (or potentially more) loci followed by electrophoresis in an automated DNA sequencer. We extended the original SNP-SCALE method to include (i) use of a commercial multiplex PCR kit, (ii) a four-dye system, (iii) much-reduced (2-μL) reaction volumes, (iv) drying down of template DNA before PCR, (v) use of pig-tailed primers, (vi) a PCR product weighting system, (vii) a standard optimized touchdown PCR thermocycling programme, and (viii) software (SNP-SCALE Primer Designer) that automatically designs suitable SNP-SCALE primers for a batch of loci. This new protocol was validated for different types of SNPs. The method is cost- and time-effective for medium-scale evolutionary and ecological projects involving 10s to 100s of loci.     

Short microsatellite DNA markers for the red fox (Vulpes vulpes)

Seven short microsatellite loci (< 165 bp) were characterized for red foxes for the amplification of degraded DNA extracted from historical samples. Following polymerase chain reaction (PCR) using primers developed in the domestic dog, red fox‐specific primers were designed within the flanking regions. The number of detected alleles ranged between six and 15 alleles and the expected heterozygosities ranged between 0.67 and 0.92. No deviations from Hardy–Weinberg equilibrium were detected for any of the markers.     

HLA-DQA1, AB0, and AMEL genotyping of biological material with biochips

A method for genotyping of biological material for AB0, HLA-DQA1, and AMEL loci is described. The method utilizes allele-specific SNP typing with th e help of hydrogel biochip technology. Amplified fluorescentlylabeled fragments of genes were hybridized with SNP-specific DNA probes immobilized on a biochip. The alleles of the gene in a sample were determined according to the distribution of the fluorescent signal. The minimal amount of biological material required for the assay is 100 pg of DNA. The biochip assay was used to analyze 442 DNA specimens of the Eastern Slavic population of Russia and to determine the allelic frequencies of AB0 and HLA-DQA1 loci. The feasibility of genotyping the biological traces of investigative value, such as cigarette butts, sweat traces on paper, and swabs of the lip of the glass, was demonstrated. This assay is applicable in forensic studies. The significance of identification for the used loci is 99.6%.     

Characterization of microsatellite markers for the mangrove tree Avicennia germinans L. (Avicenniaceae)

We describe 10 primers for amplification of microsatellite loci for the mangrove, Avicennia germinans. Eight loci were isolated from a DNA sample from the Pacific coast of Baja California, Mexico and two loci were isolated from a DNA sample from the Atlantic coast of Bermuda. Polymorphism was investigated in a population from the Mexican Pacific coast (n = 15) and in four samples scattered throughout the range of the species. Total number of alleles for the species ranged from two to 10. Observed heterozygosity in the Mexican Pacific coast population ranged from 0.27 to 0.60, with two loci having fixed alleles.     

Isolation of dinucleotide microsatellite loci from red‐backed salamander (Plethodon cinereus)

We isolated 13 variable dinucleotide microsatellites from red‐backed salamanders (Plethodon cinereus). After generating fragments using degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR), AC repeats were captured using biotinylated probes and streptavidin‐coated magnetic particles. Captured fragments were cloned into plasmids, screened for microsatellites with a simple PCR reaction, and select plasmids then sequenced. PCR primers were designed and optimized for robust amplification, and nine primers have been further optimized for multiplex reactions with fluorescent primers. These nine loci are variable with an average of 6.11 alleles per locus and an average heterozygosity of 0.61.     

微卫星复杂结构对分型的影响：以熊UamD116 和UamB1 为例

目的:微卫星是基因组上的短串联重复序列,具有高度多态性,表现为核心序列中重复单位的重复次数的变化,这种变化造成不同等位基因核心序列的长度不同。因此,其基因型主要依靠PCR扩增片段长度来判定。在各类研究中,人们更倾向于使用4碱基重复的微卫星以减少2碱基微卫星的stutter等问题的影响。但是4碱基微卫星核心序列结构复杂时,就会对分型的正确性产生影响,从而影响到下游分析的正确性。在很多野生动物的研究中,这一问题常常被忽略。本文以亚洲黑熊(Ursus thibetanus)的2个四碱基微卫星位点UamD116和UamB1为例,揭示内部结构对分型的影响。方法:我们选用96份亚洲黑熊样品(包括血液、肌肉组织和毛发等样品)进行微卫星分型研究,通过荧光标记的PCR扩增和毛细管电泳分型,比较了基于扩增片段长度的分型和基于序列核心结构的分型效果的差异。结果:UamD116核心序列结构除了含有多种不同的重复单位外,还在重复单位之间有碱基插入,出现单碱基T、二碱基TC和三碱基AAG插入;并在一类等位基因下游侧翼序列有1个GA缺失。基于序列结构的分型中可以将不同的等位基因分开,而在基于片段长度的分型中,容易将不同的等位基因合并为1个等位基因。在位点UamB1共发现两种类型的等位基因,在一类等位基因中出现一个3bp的插入,使等位基因之间的差异不再是4bp,而是1bp。在仅依据片段长度分型时,相差1bp的等位基因被认定为1个。此外,还有不同等位基因核心序列不同,但是二者长度完全一致。依据片段长度分型共发现8个等位基因,而经过序列分型确定的等位基因数为12个,相应地基因频率及其他遗传学参数都发生相应的改变。结论:对于核心序列结构复杂的微卫星必须通过等位基因测序来矫正片段长度分型的结果,才能得到可靠的群体遗传学结论。     

Cloning genomic sequences using long-range PCR

Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described.     

Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing

Background

HLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.

Results

We developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.

Conclusions

In this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1514-4) contains supplementary material, which is available to authorized users.