Genetic Linkage Facility

We have created a genetic linkage center at the Molecular Genetics Core Laboratory of the General Clinical Research Center. The core of this facility is an automated fluorescent DNA sequencer as the core of this facility integrated into a network of computers that are responsible for data management, genotype analysis and linkage analysis. With this facility, we can produce and analyze genotypes with much greater efficiency when compared to standard techniques utilizing radioactivity for pattern detection, manual band calling and sizing, and manual data entry and management. We have incorporated a number of procedures that increase throughput and minimize error in linkage analysis. These include; (1) multiplexing short tandem repeat polymorphisms (STRPs) marker amplification, (2) dual dye fluorescent detection of the STRP pattern, (3) computer assisted band calling and sizing, and (4) creation of a database that is responsible for sample tracking, sample information management, pedigree information and the creation of files for the linkage analysis programs. The different steps of the linkage analysis are performed on computers connected via a high speed network that provides easy access to mass storage disks for storage and retrieval of image files, as well as access to the sample information database.

Our facility utilizes the LI-COR fluorescent DNA sequencer. We also have several Pentium based computer workstations running applications used for STRP pattern analysis and data management. All computers are networked using ethernet connections. The following illustrates the sequence of steps we use for genetic linkage analysis. Linkage analysis begins with the identification of either populations, families or sib pairs that are segregating a genetic disorder. We have set us a number of consultants to help identify the best population to utilize as well as the necessary numbers. From each individual to be analyzed, blood or tissue samples are obtained for DNA extraction. When these samples are received by our facility, each is assigned an unique identification number which is entered into the sample database. This number is used as a reference for all information associated with the sample. Also entered into this database is the study number, pedigree number, pedigree information (e.g. identification of mother, father and siblings using their identification number), affection status, contact information, how the sample was stored (e.g. DNA, lymphocytes, lymphoblastoid cell line), the location of the samples in storage and any other pertinent information. Pedigree information and affection status are necessary for the final linkage analysis.

For the production of the STRP patterns, the first step is to determine the concentration of DNA and create a working solution of 20 ng/µl. A typical mapping study will require the analysis of numerous genotypes from many individuals, both for the initial genetic screen and subsequent fine mapping analysis. To accommodate the multiple PCR amplifications that will be necessary, a 96 well master plate is created that contains 50 to 100 µl of a working genomic DNA solution from 96 different individual to be analyzed. The DNA samples in the master plate are arranged in the wells so that when the samples are loaded into the electrophoretic gel and the STRP patterns analyzed, individuals in nuclear families are grouped in adjacent lanes. This arrangement aids in the scoring of the bands in the STRP pattern. The master plate is stored refrigerated in a plastic bag to reduce evaporation between amplification reactions.

PCR amplification for STRP production is also done in 96-well microtiter plates. STRPs are amplified in multiplexed primer sets to reduce the total number of PCR amplifications. For a complete genome screen, the Research Genetics 8A set of STRP autosomal markers have been divided into multiplexed reaction sets. The marker to marker distance averages 25 cM. The majority of the multiplexed sets contain 3 primer pairs. For each STRP primer pair, the forward primer has a modified M13 sequence added to the 5' end and included in each PCR reaction is an M13 sequencing primer that has an IR fluorescent molecule conjugated directly to the primer. For a given reaction, either the short wavelength or long wavelength fluorescent dye, conjugated to the M13 primer, is added as the sole primer containing the fluorescent dye. In the initial cycles of the amplification reaction, the forward and reverse primers for each STRP amplify the product (Figure A). In further cycles, the M13 primer will take the place of the forward primer (Figure B) to create an amplified product containing the fluorescent dye that can be detected by the LI-COR sequencer (Figure C). In setting up the amplification reactions, 1 µl of the genomic DNA is pipetted from the master plate into the 96 well amplification plate using an 8 barrel pipettor. To this, 4 µl of the reaction cocktail is added to each well including up to 8 amplification primers (4 sets of 2 primers), the M13 labeled sequencing primer, and the PCR amplification components. After amplification, 2.5 µl of stop buffer containing formamide is added. Before electrophoresis, the sample is heated to 95˚C for 2 minutes and snap cooled on ice to denature the DNA.

We have also divided the Weber 9 set of markers into multiplexed sets. The average marker to marker distance for this marker set is 10 cM. Unlike the 8A set of tailed set of markers, these primer pairs have a fluorescent label attached directly to the forward primer.

For dual-dye genotyping, the LI-COR 4200S DNA sequencer is used for separation and visualization of the STRP banding pattern. STRP reaction products are electrophoresed using 33 cm gels, 0.4 mm thick containing a 6.0 % Long Ranger gel solution. The gel is run at 1200 V constant voltage and the temperature maintained at 47˚C. Samples are loaded on the gel using an 8 barrel Hamilton sample loading syringe with 64 sample lanes and 3 size marker lanes loaded for each run. For sample loading, 1.0 µl from the high wavelength and low wavelength fluorophore containing reactions are loaded into each lane. With the LI-COR 4200 dual dye system, we can analyze 384 genotypes per run when three markers are multiplexed together in each reaction. Electrophoresis requires 2 to 3 hours with the resulting two TIFF images (one from each wavelength) produced in 16-bit format. The 16-bit format images provides a wide dynamic range when compared to 8-bit format images, allowing both strong and weak STRP patterns to be analyzed from the same multiplexed STRP run. The resulting TIFF image files are transferred and stored on a 9 Gb disk drive. When working on a project, we will run up to 3 gels in a day, using the same gel 2 to 3 times before recasting a new gel allowing one DNA sequencer to produce 1150 genotypes per day. All images are archived on recordable CD-ROM disks.

Band analysis is accomplished on a Pentium-based computer using Gene ImagIR software. This software is responsible for both automated band calling and sizing. First, the TIFF image is imported from the storage drive into the computer running Gene ImageIR. Each marker pattern is analyzed separately, though multiple marker patterns will be present on the initial TIFF image. A software template is constructed with Gene ImagIR that identifies each sample by its unique identification number, and the three lanes that contain size markers. This template can be used for all subsequent runs that amplify DNA samples from the same master plate. The sample lanes and the marker lanes are identified by the operator and the band calling software then marks a maximum of two bands (alleles) in each sample lane. After user approval of the computer called bands, which includes the binning of the same size bands for all lanes, the bin marker sizes are imported into the database along with the sample identification numbers. In the database, allele information (marker sizes) is combined with pedigree information using the unique identification number as the common relationship between the different sets of data. Files are created from this information for use by the linkage analysis programs including LINKAGE and GeneHunter.

Connection of the imaging computers, database and analysis computers using high speed networking is an important aspect of this facility. This networking of the database and storage drives allows us to manage all aspects of data collection with maximum efficiency, with a minimum amount of error. Our facility should provide all of the techniques needed for a complete linkage analysis of your population.