Analysis of Polymorphic Microsatellite DNA Sequences by PCR Amplification of Paraffin-embedded Tissue

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

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CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems

Microorganisms ◽

10.3390/microorganisms9040816 ◽

2021 ◽

Vol 9 (4) ◽

pp. 816

Author(s):

Matthew G. Links ◽

Tim J. Dumonceaux ◽

E. Luke McCarthy ◽

Sean M. Hemmingsen ◽

Edward Topp ◽

...

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Dna Sequences ◽

Pcr Amplification ◽

Shotgun Sequencing ◽

Natural Ecosystems ◽

Gene Markers ◽

Microbial Profile ◽

Microbial Ecosystems ◽

Pcr Targeting

Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.

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A primer design strategy for PCR amplification of GC-rich DNA sequences

Clinical Biochemistry ◽

10.1016/j.clinbiochem.2011.02.001 ◽

2011 ◽

Vol 44 (8-9) ◽

pp. 692-698 ◽

Cited By ~ 5

Author(s):

Li-Yan Li ◽

Qiang Li ◽

Yan-Hong Yu ◽

Mei Zhong ◽

Lei Yang ◽

...

Keyword(s):

Dna Sequences ◽

Pcr Amplification ◽

Design Strategy ◽

Primer Design

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ISOLASI FRAGMEN GEN PENYANDI PUTRESIN N-METILTRANSFERASE DAN QUINOLINAT FOSFORIBOSILTRANSFERASE ASAL TEMBAKAU LOKAL TEMANGGUNG (Nicotiana tabacum)

Jurnal Penelitian Tanaman Industri ◽

10.21082/jlittri.v17n3.2011.109-117 ◽

2020 ◽

Vol 17 (3) ◽

pp. 109 ◽

Cited By ~ 1

Author(s):

SESANTI BASUKI ◽

NURHAJATI AA MATTJIK ◽

SUWARSO SUWARSO ◽

DESTA WIRNAS ◽

SUDARSONO SUDARSONO

Keyword(s):

Dna Sequences ◽

Pcr Amplification ◽

Data Bank ◽

Degenerate Primer ◽

Gene Encoding ◽

Methyl Transferase ◽

Dna Database ◽

Genes Encoding ◽

Tobacco Cultivar ◽

Dna Template

ABSTRAKUpaya untuk menurunkan kandungan nikotin merupakan salah satuprioritas utama penelitian tembakau. Nikotin adalah senyawa alkaloidutama berpotensi dikonversi menjadi senyawa nor-nikotin yang bersifatkarsinogen. Gen PMT sebagai penyandi enzim putresin n-metiltransferase(PMT) dan gen QPT - penyandi enzim quinolinat fosforibosiltransferase(QPT) merupakan dua gen kunci yang berperan penting pada proses bio-sintesis nikotin. Penelitian ini bertujuan untuk mengisolasi potongan genPMT dan QPT asal tembakau lokal Indonesia, mengkarakterisasi danmenganalisis runutan DNA-nya. Tahapan penelitian dimulai dengan me-rancang primer degenerate berdasarkan informasi yang ada di pangkalandata Bank Gen NCBI (National Centre for Biotechnology Information),mengamplifikasi PCR menggunakan templat DNA genomik tembakaulokal cv. Sindoro1, mengklon potongan DNA hasil PCR dan menentukanrunutan DNA-nya. Hasil penelitian menunjukkan dari dua belas pasangprimer degenerate yang dirancang, hanya dua pasang primer yang meng-hasilkan potongan DNA hasil amplifikasi PCR, yaitu pasangan primerPMt-7 (F & R) untuk gen PMT dan primer QPt-3 (F & R) untuk gen QPT.Setelah dilakukan penentuan runutan DNA-nya, amplikon yang didapatdari hasil PCR dengan pasangan primer PMt-7 sebesar 1418 bp, sedangkanuntuk primer QPt-3 sebesar 205 bp. Runutan DNA gen PMT dan gen QPTasal tembakau lokal cv. Sindoro1 mempunyai tingkat kesamaan yang ting-gi dengan gen PMT dan gen QPT asal tembakau lainnya yang ada dipangkalan data Bank Gen NCBI.Kata kunci : Gen PMT, gen QPT, lintasan biosintesis nikotin, perunutanDNA, amplifikasi PCR, primer degenerateABSTRACTIsolation of Genes encoding Putrescine N-Methyl-transferase and Quinolinat Phosphoribosyl transferasederived from Temanggung Tobacco Cultivar (Nicotianatabacum)Reduction of nicotine content is one of the major objective intobacco research. Nicotine is the main alcaloid compound that potentiallycould be converted into a carcinogenic compound (nor-nicotine). The PMTgene encoding putrescine N-methyl transferase (PMT) and the QPT gene -encoding quinolinate phosphoribosyl transferase (QPT) are the two keyenzymes involved in nicotine biosynthesis. The objectives of this researchwere to isolate PMT and QPT gene fragments originated from Indonesianlocal tobacco, to characterize, and to analyze their DNA sequences. Theresearch activities included: degenerate primer design based oninformation available in the GenBank DNA Database NCBI (NationalCentre for Biotechnology Information), PCR amplification usingdegenerate primer and genomic DNA template of a local tobacco cv.Sindoro1, clone the PCR amplified products, and determine their DNAnucleotide sequences. Results of the experiment indicated that from 12degenerate primer pairs synthesized, only two were able to yield positivePCR amplified products. These primer pairs were PMt-7 (F & R primers)for PMT and QPt-3 (F & R primers) for QPT. After DNA sequencing, theamplified DNA product amplified using PMt-7 degenerate primer pairswere 1418 bp, while that using QPt-3 primer pairs were only 205 bp.Nucleotide sequences of PMT or QPT gene fragments originated fromlocal tobacco cv. Sindoro1 showed a high nucleotide sequences identity ascompared to that of the respective genes from other tobacco species thatwere available in the GenBank DNA Database NCBI.Key words: PMT gene, QPT gene, nicotine biosynthetic pathways, DNAsequencing, PCR amplification, degenerate primer

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Polymorphic microsatellite DNA markers for the white-breasted thrasher, Ramphocinclus brachyurus

Molecular Ecology Notes ◽

10.1111/j.1471-8286.2006.01375.x ◽

2006 ◽

Vol 6 (3) ◽

pp. 862-864 ◽

Cited By ~ 2

Author(s):

LI JIN ◽

HELEN JANE TEMPLE ◽

JOSEPH IVAN HOFFMAN ◽

SHENGJIANG TAN ◽

WILLIAM AMOS

Keyword(s):

Dna Markers ◽

Microsatellite Dna ◽

Microsatellite Dna Markers ◽

Polymorphic Microsatellite

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New Male Specific Markers for Hop and Application in Breeding Program

Scientific Reports ◽

10.1038/s41598-019-50400-z ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Andreja Čerenak ◽

Zala Kolenc ◽

Petra Sehur ◽

Simon P. Whittock ◽

Anthony Koutoulis ◽

...

Keyword(s):

Multiplex Pcr ◽

Dna Sequences ◽

Low Cost ◽

Pcr Amplification ◽

Breeding Program ◽

Phenotypic Data ◽

World Collection ◽

Crude Sample ◽

Male Sex ◽

Male Specific

Abstract Male specific DNA sequences were selected from a Diversity Arrays Technology (DArT) mapping study to evaluate their suitability for determination of the sex phenotype among young seedlings in a hop (Humulus lupulus L.) breeding program. Ten male specific DArT markers showed complete linkage with male sex phenotype in three crossing families. Following optimization, four were successfully converted into PCR markers and a multiplex PCR approach for their use was developed. Among 197 plants (97 from the world collection; 100 from three segregating families), 94–100% positive correlation with sex phenotypic data was achieved for the single PCR amplification, whereas the multiplex approach showed 100% correlation. To develop a fast and low-cost method, crude sample multiplex PCR was evaluated in 253 progenies from 14 segregating populations without losing accuracy. The study describes, for the first time, the routine application of molecular markers linked to male sex in an intensive Slovenian hop breeding program. The methods described could be employed for screening of sex at the seedling stage in other hop programs worldwide, thereby saving resources for desirable female plants.

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