Gene Sequencing: Introduction, and Frequently Asked Questions (FAQs)

Introduction of Gene Sequencing

Deoxyribonucleic acid (DNA) sequencing is the process of finding out the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology (Sanger/Next-generation sequencing) that is used to find out the order of the four bases i.e. adenine, guanine, cytosine, and thymine.

Cryptococcus Sequencing

Cryptococcus Sequencing
Fig. Cryptococcus Sequencing

Oxford Nanopore’s MinION Sequencing Device

Oxford Nanopore's MinION Sequencing Device
Fig. Oxford Nanopore’s MinION Sequencing Device

Frequently Asked Questions (FAQs) in Gene Sequencing

  • If you have PCR products and you are requesting gene sequencing following information should be submitted-
  • The size of primer used ( e.g. <500 bp).
  • If you have performed electrophoresis, mention the status of bands (1 band, 2 bands, 3 bands, or more).
  • Also write about the type of sequencing in which you are interested and the test charge depends on this too (forward sequencing, reverse sequencing, or both).
  • You can also request sample purification.
  • You can also order either Sanger sequencing or next-generation sequencing.
  • Multiple bands of PCR products are bad indicators for gene sequencing.

Keynotes

  • Sanger sequencing is an old method even though it is considered the “gold standard” sequencing method for validating the sequence of specific genes.
  • DNA is the building block of genes.
  • Oxford Nanopore’s MinION Sequencing Device is a real-time device for DNA and RNA sequencing.
  • MinIon is also useful for Microbial Whole Genome Sequencing Applications.
  • In general, larger DNA concentrations result in stronger signal intensities; nevertheless, sequencing issues can arise if the optimal (as shown in the table) concentrations are exceeded.
  • The reason for poor or no sequence is due to-
  1. lacking template or primer
  2. Addition of too much primer
  3. Poor quality template
  • The total volume of a sample in gene sequencing should be equal to 8µl.
  • The sequencing also depends on the length and purity of the PCR product/template.
  • The recommended quantities of Template DNA for sequencing are as follows-
TemplateQuantity
PCR product (100-200 bp)1-3 ng
200-500 bp3-10 ng
500-1000 bp5-20 ng
1000-2000 bp10-40 ng
>2000 bp40-100 ng
Single-stranded50-100 ng
Double-stranded (Plasmid DNA)250-500 ng
Cosmid, BAC
Table: Quantities of Template DNA for sequencing

Further Reading

  • https://www.nature.com/articles/s41598-018-29334-5
  • https://nanoporetech.com/products/minion
  • https://osa.stonybrookmedicine.edu/research-core-facilities/dna-sequencing/faqs
  • https://www.biotechniques.com/pcr-sequencing/what-is-dna-sequencing-answering-some-of-the-most-frequently-asked-questions/
  • https://web.genewiz.com/wgs-faq

192 thoughts on “Gene Sequencing: Introduction, and Frequently Asked Questions (FAQs)”

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  92. 2024娛樂城的創新趨勢

    隨著2024年的到來,娛樂城業界正經歷著一場革命性的變遷。這一年,娛樂城不僅僅是賭博和娛樂的代名詞,更成為了科技創新和用戶體驗的集大成者。

    首先,2024年的娛樂城極大地融合了最新的技術。增強現實(AR)和虛擬現實(VR)技術的引入,為玩家提供了沉浸式的賭博體驗。這種全新的遊戲方式不僅帶來視覺上的震撼,還為玩家創造了一種置身於真實賭場的感覺,而實際上他們可能只是坐在家中的沙發上。

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    Vote justification forms provide transparency in decision-making. Members articulate their reasoning, fostering a culture of openness and ensuring that decisions are well-founded and understood by the academic community.

    2. How can absentee ballots be cast?
    To accommodate absentee voting, PTCs may implement secure electronic methods or designated proxy voters. This ensures that faculty members who cannot physically attend meetings can still contribute to decision-making processes.

    3. How will additional members of PTCs be elected in departments with fewer than four tenured faculty members?
    In smaller departments, creative solutions like rotating roles or involving faculty from related disciplines can be explored. Flexibility in election procedures ensures representation even in departments with fewer tenured faculty members.

    4. Can a faculty member on OCSA or FML serve on a PTC?
    Faculty members involved in other committees like the Organization of Committee on Student Affairs (OCSA) or Family and Medical Leave (FML) can serve on a PTC, but potential conflicts of interest should be carefully considered and managed.

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    A positive vote typically indicates approval or agreement, while a negative vote signifies disapproval or disagreement. Clear definitions and guidelines within each PTC help members interpret and cast their votes accurately.

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    A quorum, the minimum number of members required for a valid meeting, is essential for decision-making. Specific rules about quorum size are usually outlined in the PTC’s governing documents.

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