Bacterial Artificial Chromosome (BAC) Fingerprinting

Figure 1: Electropherogram of BAC fingerprinting data

Fingerprinting of large insert genomic fragment libraries, also known as BAC (bacterial artificial chromosomes) clones leads to the construction of a genome-wide physical map. These maps are critical to genome sequencing, positional cloning and understanding the relative organization of genes and markers. When BAC libraries are arranged into maps that reflect the DNA sequence in a chromosome, they provide maximal information and utility.

Electropherogram of BAC fingerprinting data (Figure 1) analyzed on an Applied Biosystems Genetic Analyzer. Data was analyzed by sizing the restriction enzyme digested fragments with GeneMapper Software.

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Experimental Workflow: Find the Products You Need for Every Step
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Step 1: DNA Purification

DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.

Products for Step 1: DNA Purification

View more reagents, kits and bench-top devices on invitrogen.com

BAC Plasmid DNA Purification
Purification of Plasmid DNA is a technique used to prepare DNA for downstream applications such as PCR, cloning or transfection experiments. Invitrogen's range of Plasmid DNA purification kits are designed to isolate plasmid DNA and provide DNA purity you require in the scales and format you need.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Step 2: Primer Design & PCR Amplification

Purified BAC DNA is digested with a suitable combination of restriction enzymes which feature G, A, T, or C as first base at the 3’ overhang.

Products for Step 2: Primer Design & PCR Amplification

View more reagents, kits and bench-top devices on invitrogen.com

Restriction Enzymes
Purified BAC DNA is digested with a suitable combination of restriction enzymes which feature G, A, T, or C as first base at the 3’ overhang.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Step 3: Sample Prep for Fragment Analysis Run

The restriction ends are labeled by incorporation of a fluorescently-labeled dideoxynucleotide complementary to the informatory first base at the 3’ end.

Products for Step 3: Sample Prep for Fragment Analysis Run

SNaPshot™ Multiplex System

The SNaPshot® Multiplex System is a primer extension-based method that enables multiplexing up to 10 SNPs (single nucleotide polymorphisms). Use this system to screen and confirm SNPs, assess DNA methylation, fingerprint BACs, and screen samples for scrapies susceptibility.

SNaPshot® Matrix Standard Set DS-02

The Matrix Standard Set is used to generate the "multicomponent matrix" required when analyzing (add dyes) DNA fragments for the SNaPshot® kit on the Applied Biosystems 3130, 3100, 3500 and 3730 Series Systems.

Step 4: Cleanup Sample

Denaturation of double-stranded fragments by heat and formamide and subsequent removal of free dye terminators. LIZ size standards are included into each sample for precise sizing.

Products for Step 4: Cleanup Sample

Hi-Di Formamide

Highly deionized formamide used to resuspend samples before electrokinetic injection in capillary electrophoresis systems.

BigDye XTerminator® Purification Kit

The BigDye XTerminator® Purification Kit is a fast, simple purification method for DNA sequencing reactions that removes unincorporated BigDye® terminators. No more dye blobs! Cleanup is complete in under 40 minutes and requires less than 10 minutes of labor.

GeneScan™ Size Standards

GeneScan™ Size Standards are fluorescently-labeled DNA fragments required for performing fragment analysis and genotyping applications on our capillary electrophoresis platforms.

Step 5: Capillary Electrophoresis

After performing the purification, samples are ready for analysis on an Applied Biosystems capillary electrophoresis-based genetic analyzer. During capillary electrophoresis, the products of the cycle sequencing reaction are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries toward the positive electrode.

Products for Step 5: Capillary Electrophoresis

3500 Genetic Analyzer

The 3500 8-capillary platform can run a wide variety of applications—including de novo sequencing and resequencing (mutational profiling)—as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.

3500xL Genetic Analyzer

The 3500xL 24-capillary platform can run a wide variety of applications, including de novo sequencing and resequencing (mutational profiling), as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.

3730 DNA Analyzer

The 48-capillary 3730 DNA Analyzer is the Gold Standard in medium-to-high throughput genetic analysis.

3730xl DNA Analyzer

The 96-capillary 3730xl DNA Analyzer is the Gold Standard for high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample.

3130 Genetic Analyzer

Applied Biosystems 3130 Genetic Analyzer is the latest generation of 4-capillary electrophoresis instruments for the low to medium throughput laboratories. The system offers industry-leading performance, plus sophisticated automation capabilities allowing you to save time, reduce costs and increase productivity.

3130xl Genetic Analyzer

Applied Biosystems 3130xl Genetic Analyzer is the latest generation of 16-capillary electrophoresis instruments for the medium throughput laboratories.

310 Genetic Analyzer

The ABI PRISM® 310 Genetic Analyzer is an automated single-capillary genetic analyzer designed for a wide range of sequencing and fragment analysis applications.

Step 6: Data Analysis

Labeled restriction fragments are sized and valid data are exported as Table for further use in downstream mapping applications (e.g. FBP or GenoProfiler).

Products for Step 6: Data Analysis

GeneMapper® Software

GeneMapper is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems.
Literature
Application Notes