Restriction Enzyme-based Methods Research Articles

Development of PCR primers enabling the design of flexible sticky ends for efficient concatenation of long DNA fragments.

We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing o-nitrobenzyl photo-cleavable groups with a tert-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.

Mapping Mammalian 3D Genomes by Micro-C.

3D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture (3C) assays continue to discover critical roles of genome folding in regulating nuclear functions. To interrogate the full spectrum of chromatin folding ranging from the level of nucleosomes to full chromosomes in mammals, we developed an enhanced 3C-based method called Micro-C. The protocol employs Micrococcal nuclease (MNase) to fragment the genome, which overcomes the resolution limit of restriction enzyme-based methods, enabling the estimation of contact frequencies between proximal nucleosomes. Such improvements successfully resolve the fine-scale level of chromatin folding, including enhancer-promoter or promoter-promoter interactions, genic and nucleosomal folding, and boost the signal-to-noise ratio in detecting loops and substructures underlying TADs. In this chapter, we will thoroughly discuss the details of the Micro-C protocol and critical parameters to consider for generating high-quality Micro-C maps.

A standard for near-scarless plasmid construction using reusable DNA parts

Here we report GT (Guanin/Thymine) standard (GTS) for plasmid construction under which DNA sequences are defined as two types of standard, reusable parts (fragment and barcode). We develop a technology that can efficiently add any two barcodes to two ends of any fragment without leaving scars in most cases. We can assemble up to seven such barcoded fragments into one plasmid by using one of the existing DNA assembly methods, including CLIVA, Gibson assembly, In-fusion cloning, and restriction enzyme-based methods. Plasmids constructed under GTS can be easily edited, and/or be further assembled into more complex plasmids by using standard DNA oligonucleotides (oligos). Based on 436 plasmids we constructed under GTS, the averaged accuracy of the workflow was 85.9%. GTS can also construct a library of plasmids from a set of fragments and barcodes combinatorically, which has been demonstrated to be useful for optimizing metabolic pathways.

CGT-seq: epigenome-guided de novo assembly of the core genome for divergent populations with large genome.

Genetic diversity in plants is remarkably high. Recent whole genome sequencing (WGS) of 67 rice accessions recovered 10,872 novel genes. Comparison of the genetic architecture among divergent populations or between crops and wild relatives is essential for obtaining functional components determining crucial traits. However, many major crops have gigabase-scale genomes, which are not well-suited to WGS. Existing cost-effective sequencing approaches including re-sequencing, exome-sequencing and restriction enzyme-based methods all have difficulty in obtaining long novel genomic sequences from highly divergent population with large genome size. The present study presented a reference-independent core genome targeted sequencing approach, CGT-seq, which employed epigenomic information from both active and repressive epigenetic marks to guide the assembly of the core genome mainly composed of promoter and intragenic regions. This method was relatively easily implemented, and displayed high sensitivity and specificity for capturing the core genome of bread wheat. 95% intragenic and 89% promoter region from wheat were covered by CGT-seq read. We further demonstrated in rice that CGT-seq captured hundreds of novel genes and regulatory sequences from a previously unsequenced ecotype. Together, with specific enrichment and sequencing of regions within and nearby genes, CGT-seq is a time- and resource-effective approach to profiling functionally relevant regions in sequenced and non-sequenced populations with large genomes.

\u201cSame difference\u201d: comprehensive evaluation of four DNA methylation measurement platforms

BackgroundDNA methylation in CpG context is fundamental to the epigenetic regulation of gene expression in higher eukaryotes. Changes in methylation patterns are implicated in many diseases, cellular differentiation, imprinting, and other biological processes. Techniques that enrich for biologically relevant genomic regions with high CpG content are desired, since, depending on the size of an organism’s methylome, the depth of sequencing required to cover all CpGs can be prohibitively expensive. Currently, restriction enzyme-based reduced representation bisulfite sequencing and its modified protocols are widely used to study methylation differences. Recently, Agilent Technologies, Roche NimbleGen, and Illumina have ventured to both reduce sequencing costs and capture CpGs of known biological relevance by marketing in-solution custom-capture hybridization platforms. We aimed to evaluate the similarities and differences of these four methods considering each platform targets approximately 10–13% of the human methylome.ResultsOverall, the regions covered per platform were as expected: targeted capture-based methods covered > 95% of their designed regions, whereas the restriction enzyme-based method covered > 70% of the expected fragments. While the total number of CpG loci shared by all methods was low, ~ 24% of any platform, the methylation levels of CpGs covered by all platforms were concordant. Annotation of CpG loci with genomic features revealed roughly the same proportions of feature annotations across the four platforms. Targeted capture methods comprise similar types and coverage of annotations and, relative to the targeted methods, the restriction enzyme method covers fewer promoters (~ 9%), CpG shores (~ 8%) and unannotated loci (~ 11%).ConclusionsAlthough all methods are largely consistent in terms of covered CpG loci, the commercially available capture methods result in covering nearly all CpG sites in their target regions with few off-target loci and covering similar proportions of annotated CpG loci, the restriction-based enrichment results in more off-target and unannotated CpG loci. Quality of DNA is very important for restriction-based enrichment and starting material can be low. Conversely, quality of the starting material is less important for capture methods, and at least twice the amount of starting material is required. Pricing is marginally less for restriction-based enrichment, and the number of samples that can be prepared is not restricted to the number of capture reactions a kit supports. However, the advantage of capture libraries is the ability to custom design areas of interest. The choice of the technique would be decided by the number of samples, the quality and quantity of DNA available and the biological areas of interest since comparable data are obtained from all platforms.

Demethylation and alterations in the expression level of the cell cycle-related genes as possible mechanisms in arsenic trioxide-induced cell cycle arrest in human breast cancer cells.

Arsenic trioxide (As2O3) has been used clinically as an anti-tumor agent. Its mechanisms are mostly considered to be the induction of apoptosis and cell cycle arrest. However, the detailed molecular mechanisms of its anti-cancer action through cell cycle arrest are poorly known. Furthermore, As2O3 has been shown to be a potential DNA methylation inhibitor, inducing DNA hypomethylation. We hypothesize that As2O3 may affect the expression of cell cycle regulatory genes by interfering with DNA methylation patterns. To explore this, we examined promoter methylation status of 24 cell cycle genes in breast cancer cell lines and in a normal breast tissue sample by methylation-specific polymerase chain reaction and/or restriction enzyme-based methods. Gene expression level and cell cycle distribution were quantified by real-time polymerase chain reaction and flow cytometric analyses, respectively. Our methylation analysis indicates that only promoters of RBL1 (p107), RASSF1A, and cyclin D2 were aberrantly methylated in studied breast cancer cell lines. As2O3 induced CpG island demethylation in promoter regions of these genes and restores their expression correlated with DNA methyltransferase inhibition. As2O3 also induced alterations in messenger RNA expression of several cell cycle-related genes independent of demethylation. Flow cytometric analysis revealed that the cell cycle arrest induced by As2O3 varied depending on cell lines, MCF-7 at G1 phase and both MDA-MB-231 and MDA-MB-468 cells at G2/M phase. These changes at transcriptional level of the cell cycle genes by the molecular mechanisms dependent and independent of demethylation are likely to represent the mechanisms of cell cycle redistribution in breast cancer cells, in response to As2O3 treatment.

Arsenic trioxide induces cell cycle arrest and alters DNA methylation patterns of cell cycle regulatory genes in colorectal cancer cells

AimsCell cycle dysregulation is important in tumorigenesis. Transcriptional silencing of cell cycle regulatory genes, due to DNA methylation, is a common epigenetic event in malignancies. As2O3 has been shown to induce cell cycle arrest and also to be a potential hypomethylating agent. Our study aimed to investigate DNA methylation patterns of cell cycle regulatory genes promoters, the effects of Arsenic trioxide (As2O3) on the methylated genes and cell cycle distribution in colorectal cancer (CRC) cell lines. Main methodsThe methylation-specific PCR (MSP) and/or restriction enzyme-based methods were used to study the promoter methylation patterns of 24 cell cycle regulatory genes in CRC cell lines. Gene expression level and cell cycle distribution were determined by Real-time PCR and flow cytometric analyses, respectively. Key findingsOur methylation analysis indicated that only promoters of RBL1 (p107), CHFR and p16 genes were aberrantly methylated in three cell lines. As2O3 significantly decreased DNA methylation in promoter regions of these genes and restored their expression. We found that As2O3 significantly reduced the expression of DNA methyltransferase 1 (DNMT1) and increased arsenic methyltransferase (AS3MT). Furthermore, As2O3 altered transcriptional activity of several unmethylated cell cycle regulatory genes including cyclin B1, E1, D1, GADD45A and p21. Cell cycle flow cytometry analysis showed As2O3 induced G2/M arrest in all three cell lines. SignificanceThese data suggest that demethylation and alteration in the expression level of the cell cycle-related genes may be possible mechanisms in As2O3-induced cell cycle arrest in colorectal cancer cells.

The applications of thermostable ligase chain reaction in facilitating DNA recombination

The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, "Helper", to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.

RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science.

Enhanced expression of bioactive recombinant VEGF-111 with insertion of intronic sequence in mammalian cell lines.

Among all VEGF-A isoforms, VEGF-111 is particularly important in molecular biology research owing to its potent angiogenic properties and its remarkable resistance to proteolysis. These features make it a potential candidate for therapeutic use in ischemic diseases. VEGF-111 is not expressed in normal cells, but expression is induced by UV-B irradiation and exposure to genotoxic agents. Here, to increase expression at the transcriptional and translational levels, we synthesized and cloned recombinant VEGF-111 cDNA. Two fragments encoding exons 1-4 and intron 4/5 plus exon 8a were amplified and cloned into the pBud.CE4.1 vector using a class IIs restriction enzyme-based method. The expression of VEGF-111 in CHO-dhfr - and HEK 293 cell lines was evaluated with real-time PCR, dot blotting, and ELISA. VEGF expression was increased about 10- and 18-fold in transfected CHO-dhfr - and HEK 293 cells, respectively. Dot blotting and ELISA confirmed successful production of VEGF-111 in both cell lines.

Construction, imaging, and analysis of FRET-based tension sensors in living cells.

Due to an increased appreciation for the importance of mechanical stimuli in many biological contexts, an interest in measuring the forces experienced by specific proteins in living cells has recently emerged. The development and use of Förster resonance energy transfer (FRET)-based molecular tension sensors has enabled these types of studies and led to important insights into the mechanisms those cells utilize to probe and respond to the mechanical nature of their surrounding environment. The process for creating and utilizing FRET-based tension sensors can be divided into three main parts: construction, imaging, and analysis. First we review several methods for the construction of genetically encoded FRET-based tension sensors, including restriction enzyme-based methods as well as the more recently developed overlap extension or Gibson Assembly protocols. Next, we discuss the intricacies associated with imaging tension sensors, including optimizing imaging parameters as well as common techniques for estimating artifacts within standard imaging systems. Then, we detail the analysis of such data and describe how to extract useful information from a FRET experiment. Finally, we provide a discussion on identifying and correcting common artifacts in the imaging of FRET-based tension sensors.

Comparative insight into expression of recombinant human VEGF111b, a newly identified anti-angiogenic isoform, in eukaryotic cell lines

VEGF-A is a critical growth factor in tumor growth and progression. Two families of VEGF-A isoforms are produced through alternative splicing including VEGFxxx pro-angiogenic and VEGFxxxb anti-angiogenic isoforms. VEGF111b is a new member of the VEGFxxxb family that is induced by mitomycin C and doesn't express in normal conditions. The potent anti-angiogenic properties of VEGF-111b and its remarkable resistance to proteolysis make it an interesting alternative candidate for therapeutic use in all types of cancers. Here, the recombinant VEGF-111b cDNA with insertion of intronic sequence was constructed by using a class IIs restriction enzyme-based method. The recombinant pBud-VEGF111b was transfected into CHO dhfr− and HEK 293 cell lines which are currently the standard hosts for the production of candidate therapeutic proteins. Then, the VEGF-111b expression was evaluated in two cell lines using the Real-time PCR. The production of VEGF-111b protein was also investigated here by dot blotting. The VEGF expression was increased about 109 and 185-folds in transfected CHO-dhfr− and HEK 293 cells, respectively, in comparison with the un-transfected cells. Dot blotting approach confirmed that both cell lines have successfully produced the VEGF-111b protein.

Modified Restriction Enzyme-based Detection of EGFR Mutations in Lung Cancer

背景与目的表皮生长因子受体(epidermal growth factor receptor, EGFR)基因突变是肺癌靶向药物疗效的可靠预测指标，因此基因突变的检测具有非常重要的临床意义。本研究建立使用常规实验仪器、高灵敏度、简便的检测表皮生长因子受体突变的方法，以利于临床中快速的检测EGFR基因突变。方法采用改良的内切酶法富集法检测251例肺腺癌DNA标本中EGFR基因外显子19缺失突变和21(L858R)点突变，并与直接测序进行比较。利用混合突变/野生型EGFR基因的细胞系测定改良方法的灵敏度。结果在251例腺癌标本DNA中使用测序法检测出EGFR外显子19突变46例、外显子21突变26例。采用改良的突变体富集法检另外测出外显子19突变78例、外显子21突变57例，总突变率53.8%。灵敏度检测显示对于外显子19和21，新方法的检测灵敏度达0.5%。结论本方法具有简便、经济、灵敏度高等特点，便于临床快速筛查非小细胞肺癌病理组织中的EGFR基因突变。

Aberrant Epigenetic and Genetic Marks Are Seen in Myelodysplastic Leukocytes and Reveal Dock4 as a Candidate Pathogenic Gene on Chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

Recombinant DNA Technologies for Construction of Precisely Designed Transgene Constructs

Genetically modified animals have been used as models in broad range of studies including pharmaceutical biology. Designing and construction of transgene constructs are the first indispensable task in generating model animals. In addition to the classical restriction enzyme-based method, still holds some advantages in generating precise constructs, site-specific recombinase-based and homologous recombination-based DNA engineering strategies (e.g. Gateway and Red/ET recombineering, respectively) have been developed and widely used for vector construction or BAC modification. In this review, the three construction methods are described and their applications are discussed such as tandem assemblies of multiple components and modification of large DNA molecules. Combinational use of these E. coli-based recombinant DNA technologies enables the generation of precisely designed vectors useful for desired genome modification for future analyses.

Limitations in Molecular Detection of Lymph Node Micrometastasis From Colorectal Cancer

Colorectal cancer patients with lymph node metastasis have a shorter survival and may require adjuvant therapy after surgery of the primary tumor. It is supposed that a more reliable diagnosis can be achieved using tumor-specific DNA mutations for the detection of metastasizing cells. To design a practical approach for a molecular diagnosis of micrometastasis, we applied direct DNA sequencing to screen 48 early stage colorectal carcinomas for the most frequent mutations of the KRAS, P53, and APC tumor genes. KRAS mutations were detected as frequently as described earlier. In contrast, the frequency of P53 and APC hot spot mutations was unexpectedly low, compared with previous studies using other screening methods or including advanced tumor stages. Not more than 31% of early stage tumors showed a mutation in at least 1 of the selected hot spot codons. Applying mutant-enriched polymerase chain reaction (PCR), the mutation of the primary tumor was detected in lymph node DNA from 2 of the KRAS-positive patients. In 1 patient, the result was not verified by subtractive iterative PCR, a principally different molecular method with high sensitivity and specificity. Our data suggest that screening for suitable markers for a molecular detection of occult lymph node metastasis cannot be restricted to small-sized hot spot regions of a few tumor genes and possibly must include tumor-specific epigenetic changes. Furthermore, restriction enzyme-based methods such as mutant-enriched PCR are not suitable to detect any mutation with equal efficiency and they should be carefully controlled to avoid false-positive detection of marker mutations in lymph node DNA.

Restriction Enzyme-based Method for Transforming Growth Factor-β1 Genotyping: Nonisotopic Detection of Polymorphisms in Codons 10 and 25 and the 5′-Flanking Region

Transforming growth factor-β1 (TGF-β1) modulates a wide spectrum of biological activities, including cell proliferation, differentiation, and adhesion as well as extracellular matrix formation. The involvement of TGF-β1 in inflammation and tissue repair has been established in the past few years, suggesting a crucial role for this cytokine in pathologies of the cardiovascular system, kidney (chronic allograft rejection in kidney transplantation, glomerulonephritis, and diabetic nephropathy), bones (osteoporosis), skin (systemic sclerosis), lung (idiopathic lung fibrosis and asthma), and liver (cirrhosis). The human TGF-β1 gene (TGFB1) , located on chromosome 19q13, contains seven exons that give rise to a precursor protein of 390 amino acids (1), which is proteolytically processed to generate the mature protein of 112 amino acids. The promoter for TGF-β1 contains two major sites for the initiation of transcription and multiple regulatory motifs, but it lacks the typical TATA or CAAT boxes (2). Seven TGFB1 polymorphisms recently have been reported as single-stranded conformational polymorphisms and applied to a multicenter study population, the Etude Cas-Temoin de L’Infarctus du Myocarde (ECTIM) study (3). Three of the seven allelic variations were localized in the 5′-flanking region of the TGFB1 gene (at positions −988, −800, and −509), three were in the coding region (codons 10, 25, and 263), and an insertion was also described in the 5′-untranslated region at position +72. The analysis of candidate genes and/or polymorphisms has been widely applied in clinical genetics to study human diseases. A rapid and easy method for genotyping is essential in such studies, in which large numbers of cases and controls are needed. The ECTIM study used allele-specific oligonucleotides (ASOs). After amplification by PCR, products were blotted into a nylon membrane and hybridized with an ASO probe. The major disadvantage of this method is that it its time-consuming and requires …