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Sanger Sequencing

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...

Mutations

Mutations

Mutations

Mutations

Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
Chromosomal Alterations Are Large-Scale Mutations
While point mutations are changes in a single nucleotide in...

CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

Spontaneous and Induced Mutations

Spontaneous and Induced Mutations

Spontaneous mutations arise infrequently during DNA replication due to errors in the process. A key factor behind these errors is tautomeric shifts in nitrogenous bases, where bases transition from keto to enol forms or amino to imino forms. This shift can alter base-pairing rules, leading to mutations. Additionally, reactive oxygen species (ROS) arising from aerobic metabolism can damage DNA, resulting in depurination (loss of a purine base) or depyrimidination (loss of a pyrimidine base).

CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

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Decoding Sanger Sequencing Chromatograms from CRISPR-Induced Mutations.

Xianrong Xie¹, Xingliang Ma¹, Yao-Guang Liu²

¹College of Life Sciences, South China Agricultural University, Guangzhou, China.

Methods in Molecular Biology (Clifton, N.J.)

|January 6, 2019

Summary

This summary is machine-generated.

CRISPR genome editing frequently causes non-chimeric mutations. A new tool, DSDecodeM, decodes Sanger sequencing chromatograms for efficient genotyping of these CRISPR-induced mutants.

Keywords:

CRISPR DSD DSDecodeM Decoding Genome editing Sanger sequencing Superimposed chromatogram

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Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

Published on: August 24, 2017

Area of Science:

Molecular Biology
Genetics
Bioinformatics

Background:

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated genome editing is a powerful tool for genetic modification.
A significant proportion of CRISPR-induced mutations in diploid organisms are non-chimeric, presenting challenges in genotyping.
Direct Sanger sequencing of PCR amplicons with heterozygous or homozygous mutations results in superimposed chromatograms with overlapping peaks.

Purpose of the Study:

To introduce a novel strategy, Degenerate Sequence Decoding (DSD), for analyzing Sanger sequencing chromatograms.
To present DSDecodeM, an automated web-based tool designed for decoding chromatograms from various CRISPR-induced mutation types.
To enhance the efficiency and accuracy of genotyping CRISPR-edited organisms.

Main Methods:

Development of the Degenerate Sequence Decoding (DSD) strategy.
Implementation of DSD into an automatic web-based tool named DSDecodeM.
Application of DSDecodeM for analyzing Sanger sequencing data from CRISPR-induced mutants.

Main Results:

The DSD strategy effectively decodes superimposed sequencing chromatograms.
DSDecodeM accurately identifies and differentiates various non-chimeric mutation types (biallelic, homozygous, heterozygous).
The tool simplifies and accelerates the genotyping process for CRISPR-generated mutants.

Conclusions:

DSDecodeM provides a convenient and versatile solution for genotyping CRISPR-induced mutants.
This tool significantly facilitates the workflow for researchers working with genome editing technologies.
Automated chromatogram decoding improves the overall efficiency of genetic engineering studies.