Kemajuan Analisis DNA Forensik... - Generasi Peneliti
Forensic DNA analysis telah menjadi salah satu alat paling kuat dalam sistem peradilan pidana. Penggunaan DNA untuk mengidentifikasi individu ...
qPCR ( quantitative Polymerase Chain Reaction)
Proses DNA typing melibatkan beberapa langkah utama: pengumpulan sampel, ekstraksi DNA, kuantifikasi DNA, amplifikasi DNA, analisis hasil, dan interpretasi data. Langkah pertama adalah mengumpulkan sampel biologis dari tempat kejadian perkara atau individu. Sampel ini bisa berupa darah, saliva, rambut, kulit, atau cairan tubuh lainnya. DNA diekstraksi dari sel dalam sampel yang dikumpulkan. Teknik ekstraksi modern telah ditingkatkan untuk mengoptimalkan pemulihan DNA, bahkan dari sampel yang sangat terdegradasi. Setelah DNA diekstraksi, langkah selanjutnya adalah mengukur jumlah DNA yang diperoleh untuk memastikan ada cukup DNA untuk dianalisis. Metode kuantifikasi DNA telah ditingkatkan dengan menggunakan teknologi <anchor>qPCR ( quantitative Polymerase Chain Reaction)</anchor>, yang memungkinkan kuantifikasi DNA dengan presisi tinggi. Amplifikasi adalah proses memperbanyak segmen DNA tertentu untuk analisis lebih lanjut. Teknik PCR ( Polymerase Chain Reaction) adalah metode utama yang digunakan untuk amplifikasi DNA. Kemajuan dalam teknologi PCR, termasuk PCR digital dan PCR multiplex, telah meningkatkan kemampuan untuk menganalisis sampel DNA dengan jumlah minimal atau kualitas rendah. Setelah amplifikasi, produk PCR dianalisis menggunakan elektroforesis kapiler atau teknik pemisahan lainnya. Perkembangan dalam teknologi elektroforesis kapiler telah meningkatkan resolusi dan kecepatan analisis DNA. Data yang diperoleh dari analisis DNA diinterpretasikan untuk membuat profil genetik. Software analisis modern telah dikembangkan untuk membantu dalam interpretasi data yang kompleks dan untuk meminimalkan kesalahan manusia.
Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR) - ScienceDirect
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## Biomolecular Detection and Quantification

# Research paper  Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)

Author links open overlay panelAdriánRuiz-Villalbaab, Elizabethvan Pelt-Verkuilc, Quinn DGunsta, Jan MRuijtera, Maurice JBvan den Hoffa

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## Abstract

Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to Cq or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. To explore the range of input variations that occur in the normal use of the Cre assay these conditions were mimicked in a complete two-way design of template −plasmid DNA- and non-template −mouse cDNA- concentrations. These experiments showed that the frequency of the amplification of the correct product and the artifact, as well as the valid quantification of the correct product, depended on the concentration of the non-template cDNA. This finding questions the interpretation of dilution series in which template as well as non-template concentrations are simultaneously decreasing. Repetition of this cDNA concentration experiment with other templates revealed that exact reproduction qPCR experiments was affected by the time it takes to complete the pipetting of a qPCR plate. Long bench times were observed to lead to significantly more artifacts. However, the measurement of artifact-associated fluorescence can be avoided by inclusion of a small heating step after the elongation phase in the amplification protocol. Taken together, this trouble-shooting journey showed that reliability and reproducibility of qPCR experiments not only depends on standardization and reporting of the biochemistry and technical aspects but also on hitherto neglected factors as sample dilution and waiting times in the laboratory work flow.

## Graphical abstract

Highlights

- •
  The occurrence of PCR artifacts depends on template, non-template and primer concentrations in the reaction.

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  Reliability and reproducibility of qPCR experiments requires standardized technical aspects and short on-bench times.

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  Measurement of artifact-associated fluorescence can be minimized by primer design and qPCR protocol.



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## Keywords

RT-qPCR

Melting curve analysis

Reaction parameters

Artifacts

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