Molecular Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 218 Views Mar 5, 2023

Malaria molecular surveillance has great potential to support national malaria control programs (NMCPs), informing policy for its control and elimination. Here, we present a new three-day workflow for targeted resequencing of markers in 13 resistance-associated genes, histidine rich protein 2 and 3 (hrp2&3), a country (Peru)-specific 28 SNP-barcode for population genetic analysis, and apical membrane antigen 1 (ama1), using Illumina short-read sequencing technology. The assay applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. The assay can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes.

0 Q&A 1376 Views Mar 20, 2022

Spontaneous DNA damage frequently occurs on the human genome, and it could alter gene expression by inducing mutagenesis or epigenetic changes. Therefore, it is highly desired to profile DNA damage distribution on the human genome and identify the genes that are prone to DNA damage. Here, we present a novel single-cell whole-genome amplification method which employs linear-copying followed by a split-amplification scheme, to efficiently remove amplification errors and achieve accurate detection of DNA damage in individual cells. In comparison to previous methods that measure DNA damage, our method uses a next-generation sequencing platform to detect misincorporated bases derived from spontaneous DNA damage with single-cell resolution.

0 Q&A 2692 Views Nov 5, 2020

Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. Here we describe DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.

0 Q&A 4352 Views May 20, 2020
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL–associated NOTCH1 mutations either disrupt the negative regulatory region, allowing signal activation in the absence of ligand binding, or result in truncation of the C-terminal PEST domain involved in the termination of NOTCH1 signaling by proteasomal degradation. To date, retroviral transduction models have relied heavily on the overexpression of aggressively truncated variants of NOTCH1 (such as ICN1 or ΔE-NOTCH1), which result in supraphysiological levels of signaling activity and are rarely found in human T-ALL. The current protocol describes the method for mouse bone marrow isolation, hematopoietic stem and progenitor cell (HSC) enrichment, followed by retroviral transduction with an oncogenic mutant form of the NOTCH1 receptor (NOTCH1-L1601P-ΔP) that closely resembles the gain-of-function mutations most commonly found in patient samples. A hallmark of this forced expression of constitutively active NOTCH1 is a transient wave of extrathymic immature T-cell development, which precedes oncogenic transformation to T-ALL. Furthermore, this approach models leukemic transformation and progression in vivo by allowing for crosstalk between leukemia cells and the microenvironment, an aspect unaccounted for in cell-line based in vitro studies. Thus, the HSC transduction and transplantation model more faithfully recapitulates development of the human disease, providing a highly comprehensive and versatile tool for further in vivo and ex vivo functional studies.
0 Q&A 3344 Views Feb 20, 2020
Chromatin immunoprecipitation is extensively used to investigate the epigenetic profile and transcription factor binding sites in the genome. However, when the starting material is limited, the conventional ChIP-Seq approach cannot be implemented. This protocol describes a method that can be used to generate the chromatin profiles from as low as 100 human or 1,000 Drosophila cells. The method employs tagmentation to fragment the chromatin with concomitant addition of sequencing adaptors. The method generates datasets with high signal to noise ratio and can be subjected to standard tools for ChIP-Seq analysis
0 Q&A 6068 Views Aug 5, 2019
Most bacterial genomes have biased nucleotide composition, and the asymmetry is considered to be caused by a single-stranded DNA (ssDNA) deamination arising from the bacterial replication machinery. In order to evaluate the relationship experimentally, the position and frequency of ssDNA formed during replication must be verified clearly. Although many ssDNA detection technologies exist, almost all methods have been developed for eukaryotic genomes. To apply these to bacterial genomes, which harbor a smaller amount of DNA than those of eukaryotes, more efficient, new methods are required. Therefore, we developed a novel strand-specific ssDNA sequencing method, called 4S-seq, for the bacterial genome. The 4S-seq method enriches ssDNA in the extracted genomic DNA by a dsDNA-specific nuclease and implements a strand-specific library using a biotin label with a customized tag. As a result, the 4S-seq is able to calculate the ssDNA content in each strand (Watson/Crick) at each position of the genome efficiently.
0 Q&A 4132 Views Jul 5, 2019
Detecting heteroplasmies in the mitochondrial DNA (mtDNA) has been a challenge for many years. In the past, Sanger sequencing was the main option to perform this analysis, however, this method could not detect low frequency heteroplasmies. Massive Parallel Sequencing (MPS) provides the opportunity to study the mtDNA in depth, but a controlled pipeline is necessary to reliably retrieve and quantify the low frequency variants. It has been shown that differences in methods can significantly affect the number and frequency of the retrieved variants.

In this protocol, we present a method involving both wet lab and bioinformatics that allows identifying and quantifying single nucleotide variants in the full mtDNA sequence, down to a heteroplasmic load of 1.5%. For this, we set up a PCR-based amplification of the mtDNA, followed by MPS using Illumina chemistry, and variant calling with two different algorithms, mtDNA server and Mutect.

The PCR amplification is used to enrich the mitochondrial fraction, while the bioinformatic processing with two algorithms is used to discriminate the true heteroplasmies from background noise. The protocol described here allows for deep sequencing of the mitochondrial DNA in bulk DNA samples as well as single cells (both large cells such as human oocytes, and small-sized single cells such as human embryonic stem cells) with minor modifications to the protocol.
0 Q&A 5180 Views May 5, 2019
Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a “bait” DNA double-stranded break (DSB) to capture genome-wide “prey” DNA DSBs. Here, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. Compared with LAM-HTGTS, iHTGTS is lower cost and has much higher sensitivity for off-target detection in HEK293T, K562, U2OS and HCT116 cell lines. So we believe that iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.
0 Q&A 5475 Views Feb 20, 2019
Single-cell DNA sequencing is a powerful tool to evaluate the state of heterogeneity of heterogeneous tissues like cancer in a quantitative manner that bulk sequencing can never achieve. DOP-PCR (Degenerate Oligonucleotide-Primed Polymerase Chain Reaction), MDA (Multiple Displacement Amplification), MALBAC (Multiple Annealing and Looping-Based Amplification Cycles), LIANTI (Linear Amplification via Transposon Insertion) and TnBC (Transposon Barcoded) have been the primary choices to prepare single-cell libraries. TnBC library prep method is a simple and versatile methodology, to detect copy number variations or to obtain the absolute copy numbers of genes per cell.
0 Q&A 5315 Views Feb 20, 2019
DNA methylation is a common mechanism of epigenetic regulation involved in transcriptional modulation and genome stability. With the evolution of next-generation sequencing technologies, establishing quantitative genome-wide DNA methylation profiles is becoming routine in many laboratories. However, many of these approaches take several days to accomplish and use subjective PCR methods to amplify sequencing libraries, which can induce amplification bias. Here we propose a rapid Reduced Representation Bisulfite Sequencing (rRRBS) protocol to minimize PCR amplification bias and reduce total time of multiplexed library construction. In this modified approach, the precise quantification of the final library amplification step is accomplished and monitored by qPCR, instead of using standard PCR and gel electrophoresis, to determine the appropriate number of cycles to perform. The main advantages of this rRRBS method are: i) Reduced amount of amplification enzyme used for library prep, ii) Reduced number of PCR cycles resulting in less PCR amplification bias, and iii) Preparation of quality multiplexed rRRBS libraries in only ~2 days.

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