ACGT, Inc. (CLIA Compliant)

Intact, high-quality genomic DNA (gDNA) is routinely extracted from blood, saliva, buccal swabs, soft tissues, or plants. We also perform total nuclei acid extractions where gDNA, plasmid DNA and RNA are concomitantly isolated from the same sample such as bacteria. For DNA extraction from plants, the Automated Sample Grinding System (AutoGen) is used to obtain consistent results. Extraction of gDNA from rodent tails or ears is part of our transgenic genotyping service.

DNA Extraction Service Description:
- DNA extraction
- Quantitative analysis by UV spectroscopy and PicoGreen
- Qualitative analysis by UV spectroscopy and agarose gel
- Sample concentration adjustment per customer specifications
- Aliquot of sample into tubes or plates, and shipping to any destination

We offer a complete Genotyping by DNA Sequencing for screening alleles based on nucleotide sequence. Alleles in any organism are detected by PCR amplification of the target gene and DNA sequencing of the resulting PCR product. Dilution PCR analysis or bulk PCR product subcloning and screening followed by DNA sequencing is an additional service provided for reliable analysis of single point mutations in low abundance. DNA sequencing is one of the most definitive methods available for allele identification and genotyping.

Genotyping by DNA Sequencing Service Description:
- Complete study design and support capabilities
- Design and synthesis of PCR amplification primers
- Optimization and verification of PCR amplification conditions for new and pre-existing strategies
- Isolation of genomic DNA, RNA and cDNA synthesis
- PCR amplification and DNA sequencing
- Dilution PCR analysis or bulk PCR product subcloning/screening followed by DNA sequencing
- Manual verification of automated base calls in sequence data
- Phred 20-40 analysis of sequence data
- Sequence data comparison analysis between samples and reference sequence
- Extensive troubleshooting experience and technical support
- FDA quality DNA sequence data available
- Complete GLP capabilities

ChIP-sequencing (ChIP-Seq) combines chromatin immunoprecipitation (ChIP) and massively parallel sequencing to identify those DNA sequences bound by transcription factors in vivo.

A very common form of ChIP-Seq experiment is the Histone Modification Analysis. Histone compaction of DNA is a well-known epigenetic regulator of gene expression. Post-translational modification of histones, such as histone deacetylation, is another common form of epigenetic control, and is known to be associated with gene silencing. Therefore, evaluation of histone coverage and histone modification status are valuable tools in understanding genome-wide regulation of gene expression.

We offer primarily Targeted Metagenomics services. Targeted metagenomics typically aim at smaller-scale goals, such as the 16S rRNA-based surveys for microbe identification, or acquiring sequence reads with specific protein functions, such as antibiotic resistance genes.

How it works: Total DNA is extracted from the sample, followed by any number of selected PCR reactions. The short PCR products (300 bp maximum size) are purified and are converted to Illumina libraries by the ligation of adapters. Multiplex barcoding is typically used to maximize sample processing efficiency. The libraries are analyzed using long paired-end reads to fully sequence each PCR product in the cluster. The reads are sorted, counted, and non-identical sequences are BLASTed against selected databases for sequence identification.

Epigenetic control is often mediated by methylation of cytosine to 5-methylcytosine (5-mC) in CpG islands. Methylation and/or hydroxymethylation of CpGs near promoters is associated with gene silencing, and has important consequences for gene expression by contributing to the re-modeling of chromatin via recruitment of methyl-CpG-binding domain (MBD) protein complexes and subsequent chromatin modifiers. Disease phenotypes have been shown to arise when these activities are perturbed, resulting in undesired gene expression.

DNA methylation status can be evaluated in two basic ways:
- Whole Genome Bisulfite Conversion.
- Affinity-based isolation of methylated DNA.

High quality plasmid DNA from the Mini to Giga scale is available for many applications including DNA sequencing, transfections, and DNA-mediated antibody production. Resin based methods are used to prepare highly pure plasmid DNA preparations with an option of making these endotoxin-free. Comparable services are available for BAC, lambda, and cosmid DNA.

Extraction of highly pure and intact RNA (total, mRNA, sRNA and miRNA) from a variety of samples including soft tissues (fresh and preserved), blood, saliva, cell cultures, plants, and forensics samples is available whether it from a few samples to large-scale project. Both liquid and resin based purification systems are used to extract the RNA species of interest. Concomitant extraction of genomic DNA and plasmid DNA is possible. The purified RNA is suitable for many downstream applications including cDNA synthesis including library construction, Northern blots and qPCR.

Service Description:
- RNA extraction by liquid or resin based methods
- Quantitative analysis by UV spectroscopy and RiboGreen
- Qualitative analysis by UV spectroscopy and agarose gel

Complete sequencing of transcriptomes has become an important tool in the analysis of gene expression, alternative splice sites, allele specific expression and the discovery and analysis of rare or novel transcripts. Unlike the microarray approach which analyzes the known transcripts contained on the array, with the NGS approach the entire RNA population contained in the sample is sequenced. This provides an unprecedented detail of the transcriptome organization and structure.

We create a random mutagenesis library of any desired complexity to any gene of your interest.

Service Description:
- Cloning of the randomized DNA into any vector
- Cloning into expression vectors
- Confirmation of desired mutation by DNA sequencing
- Selection of mutation frequencies depending on the application of the study

We offer Single Nucleotide Polymorphism (SNP) genotyping for large and small scale projects including SNP discovery, validation, and screening. Accurate and reproducible results are more accessible for projects of any size with the use of an established platform and optimized. SNPs from any organism can be genotyped.

SNP Genotyping Service Description:
- Use of the 7900HT Sequence Detection System from Applied Biosystems Inc. (ABI)
- SNP detection based on TaqMan probes with Assays-on-Demand (AOD) or Assays-by-Design (ABD) from ABI
- Data presented in user-friendly spreadsheets that contain raw fluorescent data and genotype call based on software analysis and experimenter verified
- All results are available in electronic and hard copy formats including publication quality graphics with genotype distributions for all samples.
- Experience with difficult to assay samples and complete assistance with data interpretation

We offer a complete Fragment Analysis of Short Tandem Repeats (STR) or microsatellite (MS) genotyping service. Use of multiple ABI 3730XL or 3730 Genetic Analyzers allows us to screen hundreds of samples per day. STR Genotyping is available for most organisms.

STR Genotyping Service Description:
- Design and synthesis of fluorescent-labeled PCR amplification primers for individual or multiplex strategies.
- Optimization of PCR amplification conditions for detecting STR and MS loci.
- PCR amplification of STR loci and separation on the ABI 3730XL or 3730 Genetic Analyzer.
- Allele size and identity determination with GeneMapper ID (V3.0) software and experimenter
verification of the data.
- Data presented in user-friendly spreadsheets that contain allele size and intensity.
- All results are available in electronic and hard copy formats including a publication quality electropherogram of each sample with a reference size ladder.
- Experience with difficult to assay samples and complete assistance with data interpretation.
- A "load-and-go" service is available if the user has prepared the fluorescently labeled PCR products and only requires analysis of these on the Genetic Analyzer.

We offer Quick Single Pass DNA Sequencing service for plasmid, PCR DNA, and large-insert templates (lambda, BAC, PAC, P1 clones).
"Load and go" (already cycled) - 2.50 per sample, $160 per plate
DNA sequencing and cleanup - $7 per sample, $420 per plate.

Direct Colony Sequencing
Instead of purified plasmid DNA, just send us bacterial colonies re-suspended in a small volume of buffer and a sequencing primer (separately or in the same tube). Our skilled professionals will extract and amplify the plasmid DNA directly from the colony, and generate sequencing data with the same exceptional level of quality as our standard sequencing service.

Full Sequence Analysis
ACGT, Inc. offers a Full Sequence Analysis service to confirm the sequences of a clone or detection of mutations or deletions. Our service is comprehensive and results in double-strand sequence data of publication quality.

High Throughput Sequencing
ACGT, Inc. offers High throughput DNA sequencing in a fully automated lab. We are capable of handling thousands of clones per day using a barcode system to accurately inventory and process DNA samples upon receipt until delivery of final data. Project tracking for each stage of sample processing can be viewed realtime online via our website.

We provide shotgun sequencing services for a variety of samples including BAC, PAC, or cosmid DNA for genomic scale projects.

You will receive assembled and based-called DNA sequences, publication-ready DNA sequences, electropherograms of each run, and all of the subclones, plasmid DNA, and internal primers generated for the project.

Based on PCR primer design, any type of mutation, such as deletions, insertions, or substitutions, can be introduced into your genes in a single or multiple sites. Any desired changes in amino acids, or deletions and insertions of restriction sites can be made into the genes of your interest.

Service Description:
- Design and synthesis of mutagenesis primers
- PCR amplification
- Confirmation of desired mutations by DNA sequencing
- Confirmation of undesired mutations by DNA sequencing
- Subcloning modified gene into fresh vector not subjected to the mutagenesis procedure or subcloning into another vector (Optional)
- Verification of vector sequence after mutagenesis by DNA sequencing (Optional)
- Delivery of purified plasmid DNA construct and its glycerol stock

We offer whole genome sequencing on NextSeq 500 platform, with PE150 reads. Sequencing costs for a bacterial genome are in the $200 range for 100X coverage; for human and similar size genomes, it is $5,200 for 35X coverage.

cDNA Cloning Service Description:
- Extraction of RNA from a variety of samples
- Full length and partial cDNA cloning
- Cloning via PCR, RT-PCR, 5' and 3' RACE
- Cloning into any desired vector
- Cloning open reading frame into any expression vector
- Customize protein expression by inclusion of epitope tags or fusion constructs such as green fluorescent protein at either end of protein

We offer a rapid, reliable semi-quantitative and quantitative PCR (qPCR) analysis of gene expression and gene copy number determination. Detection of targets with Taqman™ probes with the 7900HT Sequence Detection System (Applied Biosystems Inc.) or SYBR green is available for large or small scale projects alike. The instrument platform has high-throughput capabilities and can analyze over 1,500 samples on a daily basis.

The full service option includes RNA or DNA extraction, probe design and optimization and complete data analysis including normalization of samples with internal controls. Relative and absolute analysis of gene expression by qPCR is available for all known genes in any organism.