分子生物学

Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein and transcriptional regulator implicated in chromatin dynamics and cancer development. Several BRD4 isoforms have been detected in humans with the long isoform (BRD4-L, aa 1-1,362) playing a tumor-suppressive role and a major short isoform (BRD4-S, aa 1-722) having oncogenic activity in breast cancer development. In vivo demonstration of the opposing functions of BRD4 protein isoforms requires development of mouse models, particularly transgenic mice conditionally expressing human BRD4-L or BRD4-S, which can be selectively induced in different mouse tissues in a spatiotemporal-specific manner. Here, we detail the procedures used to genotype transgenic mouse strains developed to define the effects of conditional human BRD4 isoform expression on polyomavirus middle T antigen (PyMT)-induced mouse mammary tumor growth, and the key steps for Western blot detection of BRD4 protein isoforms in those tumors and in cultured cells. With this protocol as a guide, interpretation of BRD4 isoform functions becomes more feasible and expandable to various biological settings. Adequate tracking of BRD4 isoform distributions in vivo and in vitro is key to understanding their biological roles, as well as avoiding misinterpretation of their functions due to improper use of experimental procedures that fail to detect their spatial and temporal distributions.

Graphic abstract:

Association mapping is the process of linking phenotypes with genotypes. In genome wide association studies (GWAS), individuals are first genotyped using microarrays or by aligning sequenced reads to reference genomes. However, both these approaches rely on reference genomes which limits their application to organisms with no or incomplete reference genomes. To address this, reference free association mapping methods have been developed. Here we present the protocol of an alignment free method for association studies which is based on counting k-mers in sequenced reads, testing for associations between k-mers and the phenotype of interest, and local assembly of the k-mers of statistical significance. The method can map associations of categorical phenotypes to sequence and structural variations without requiring prior sequencing of reference genomes.

Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, the inheritance of specific methylation patterns can be studied. Here, we present a protocol for analyzing DNA methylation patterns through MSAP markers in potato interspecific hybrids and their parental genotypes.

Cleaved amplified polymorphic sequences (CAPS) assays are useful tools for detecting small mutations such as single nucleotide polymorphisms (SNPs) or insertion/deletions (indels) present in an amplified DNA fragment. A mutation that disrupts or creates a restriction site will prevent cleavage by a restriction enzyme, allowing discrimination of wild-type and mutant alleles. In cases where no convenient restriction site is present, a derived Cleaved Amplified Polymorphic Sequence (dCAPS) assay can be used, where mismatches in the primer are used to create a diagnostic restriction site. No special design constraints are present for a CAPS assay, but cases where CAPS assays can be used are infrequent. A dCAPS assay can be burdensome to design by hand, but it is more broadly applicable. This protocol will describe the use of the indCAPS tool for the design of CAPS and dCAPS primers. The indCAPS tool was designed to be compatible with indel alleles, which prior tools struggled with but have increased importance since the rise of CRISPR/Cas9 mutagenesis methods.

Organic anion transporting polypeptide (OATP) 1B1 is a liver-specific transport protein that plays an important role in hepatic drug disposition. It transports many drugs from the blood into the liver, including lipid-lowering statins. The c. 521 T>C polymorphism of OATP1B1 has reduced transport activity and is associated with statin-induced myopathy. Formalin-fixed paraffin-embedded (FFPE) liver tissues can be an enriched source for genotyping of this clinically significant OATP1B1 polymorphism in retrospective studies. The successfulness of genotyping using Sanger-sequencing of a PCR product from FFPE tissue relies on a successful PCR amplification using genomic DNA extracted from the FFPE tissues. Such PCR amplification is often limited by the quality of DNA extracted from the FFPE tissue. An optimized method for high-quality DNA extraction and efficient PCR amplification is highly needed in order to genotype polymorphisms such as the c. 521 T>C polymorphism using FFPE tissues. The current study established an optimized and reproducible method for a Sanger-sequencing-based genotyping method using FFPE human liver tissues that is applicable to even small FFPE tissues such as needle-core biopsy specimens.

The most commonly used mouse model in ALS preclinical research expresses multiple copies of the human SOD1 (G93A) transgene. During the course of breeding, successive generations of mice can lose copies of the transgene. Because shorter lifespan of these mice is dependent on transgene copy number, it is essential to ensure that no low-copy, and therefore longer-lived, mice are included in preclinical studies. Existing techniques for SOD1^G93A mouse genotyping are broadly based on creating a standard curve using a reference gene and deducing the relative amount of SOD1 by comparison with the standard curve. This type of technique is used in Alexander et al. (2004), Vieira et al. (2017) and Maier et al. (2018). However, it is not described in detail (see Note 1). This paper provides a detailed protocol for determining the relative copy number of the human SOD1 transgene. Briefly, the protocol involves first the extraction of high-quality genomic DNA from mouse ear tissue, creation of a genomic DNA concentration-based standard curve, and qPCR analysis of up to 88 samples at once alongside the standard curve with Gapdh as a reference gene. Analysis involves the normalization of each unknown sample using the standard curve followed by determination of the copy number of the sample relative to the cohort median. This protocol has been optimized to produce high-quality genomic DNA and consistent results, and the relative copy number cutoffs have been optimized and validated empirically by comparison of relative copy number and mouse lifespan.

Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon et al., 2013; Tunçay et al., 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the observed phenotype. In this protocol, we describe in detail an improved version of the restriction enzyme site-directed amplification PCR (RESDA-PCR) originally reported in (González-Ballester et al., 2005). The improvement includes optimization of primer combination, the choice of DNA polymerase, optimization of PCR cycle parameters, and application of direct sequencing of the PCR products. These modifications make it easier to get specific PCR products as well as speeding up subcloning steps to obtain sequencing data faster.

Diagnostic assays for pathogen identification and characterization are limited either by the number of simultaneously detectable targets, which rely on multiplexing methods, or by time constraints due to cultivation-based techniques. We recently presented a 100-plex method for human pathogen characterization to identify 75 bacterial and fungal species as well as 33 clinically relevant β-lactamases (Barišić et al., 2016). By using 16S rRNA gene sequences as barcode elements in the padlock probes, and two different fluorescence channels for species and antibiotic resistance identification, we managed to cut the number of microarray probes needed by half. Consequently, we present here the protocol of an assay with a runtime of approx. 8 h and a detection limit of 10⁵ cfu ml^-1. A total of 89% of β-lactamases and 93.7% of species were identified correctly.

The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al., 2016a and 2016b; Rose et al., 2016). This method optimizes protocols from previous publications (Palmer et al., 2005; Norström et al., 2012; Lamers et al., 2015; 2016a and 2016b; Rife et al., 2016) to produce single distinct PCR products that can be directly sequenced and includes several cost-saving and time-efficient modifications.

Herein we describe a detailed protocol for DNA virome analysis of low input human stool samples (Monaco et al., 2016). This protocol is divided into four main steps: 1) stool samples are pulverized to evenly distribute microbial matter; 2) stool is enriched for virus-like particles and DNA is extracted by phenol-chloroform; 3) purified DNA is multiple-strand displacement amplified (MDA) and fragmented; and 4) libraries are constructed and sequenced using Illumina Miseq. Subsequent sequence analysis for viral sequence identification should be sensitive but stringent.