Firma:                   Transgenomic

Modell:                HTCE 99

Kommentar:        Dokumente engl.

The Spectrumedix System is a multi application platform which can be used for:

·             Mutation / SNP + indel discovery & genotyping (TGCE)

·             Surveyor Mutation Detection and genotyping

·             SBE (single base extension) genotyping

·             Fragment Sizing

·             STR analysis

·             sequencing

·             PCR quality testing

The Spectrumedix System is a fast and reliable mutation detection system based on temperature-gradient capillary electrophoresis (TGCE). The Spectrumedix system is able to perform DNA sequencing and genotyping of fluorescently labeled DNA fragments. Both DNA sequencing and Genotyping are able to be performed on the sample plate and this is automated so that uninterrupted sequencing and genotyping can be performed over a 24 hour period. The Spectrumedix System is capable of using the Big Dye chemistry and is not restricted to this chemistry, allowing the user to use any 4-colour or 5-colour chemistry. The Spectrumedix System is available wich 24, 96 and 192 capillaries. The 24 capillary system can be upgraded to 96 or 192 capillaries anytime.

Overview of System Software and Functions

•     This system is fully automated and controlled by the SpectruMedix CheckMateTM operational software package. The software provides for run method file creation and storage, which allows you to easily create application-specific methods and recall them for future use. Data acquisition parameters are preset and once the system is configured, there aren't any run-to-run or day-to-day adjustments required, other than the occasional replacement of the capillary cartridge.

Additional Features:

•     CheckMate TM provides automated Operation for up to six sample trays.

•     The software has current monitoring capability starting with the Pre-Run stage and continuing through the end of camera Data Acquisition. These measured currents are stored in a tab-delimited text formst as a *.curfile, and are useful diagnostic tools for troubleshooting Instrument and/or chemistry issues related to electrophoresis.

•     Temperature control of the cartridge is a feature of CheckMateTm. These data sets are recorded as a *.tmp file, and can be useful for post-run diagnostics.

•     A report file is recorded for each run denoting all relevant running condition information including: voltages, solution information, camera settings, sample information, and the start/stop times for each step of the run. The report file *.rpt can be recalled after the run has finished for review.

Hardware Components

The system includes the following:

•     Multiplexed Capillary Electrophoresis Instrument – sample loading carousels and elevator, temperature control module, high voltage buffer system, laser, CCD imaging device and electronic controls

•     Instrument CPU – Computer, control cards, Gables, monitor, mouse and keyboard

•     Solution Containers – wash bottles, buffer bottles, gel syringes, and gel bottles

•     Capillary Cartridge – completely self-contained capillary array

•     UPS - uninterruptible power supply

•     Sample and Buffer Tray Holders - Six universal sample tray holders and six buffer tray holders

•     Documentation – Operators manual, software manual, chemistry protocol, and specific application notes

Principles of TGCE

Temperature gradient capillary electrophoresis (TGCE) has emerged as a powerful technology for DNA mutation and SNP discovery and screening. The method can be used to rapidly identify DNA regions containing sites of mutation or SNPs. This may then be followed by sequencing those amplicons of interest to locate the exact positions of sequence alteration.

The TGCE technique is based on the analysis of a heterozygous DNA sample with two different alleles using a capillary electrophoresis (CE) platform. It involves resolving double-stranded DNA into the heteroduplex and homoduplex moieties using a sieving gel matrix under a temporal temperature gradient (Figure 2.1). The method employs variable migration of the two moieties due to their differing melting points. During electrophoresis, a sample containing both heteroduplex and homoduplex dsDNA is injected into the capillary. As the sample migrates through the capillary, the ambient temperature is raised at a controlled rate. Due to the inherently lower stability at the base mismatch, the heteroduplex strands melt at a lower temperature than the homoduplexes. Partial melting forms a "bubble-like" structure along the sequences. The mobility of these bulkier DNA fragments is retarded in the gel matrix, resulting in separation of heteroduplexes from homoduplexes in the capillary. Using narrow temperature ramps maintains a longer retention time for each of four different conformers. In this way the different homoduplexes and heteroduplexes can be separated. Under ideal conditions, it is possible to separate the individual heteroduplexes (Figure 2.1). It is also possible to isolate all four separate species, although this occurs with considerably less frequency.

Screeninq unknown samples . A common practice is to compare the electropherogram of a test sample to that of a known homozygote belonging to the same DNA locus and score for a heterozygote (Figure 2.2). This approach has the advantages of avoiding pre-run optimization and testing amplicons with different sequences and sizes in a single sample plate or within a single capillary by using a broader temperature gradient. The degree of separation of heteroduplexes from homoduplexes is dependent on the size of the amplicon, the type of sequence variants, the location of the alteration site and the sequence adjacent to the alternation, as well as the melting profile of the sequence. A wide range of sizes of different amplicons ranging from 100 to 1,300bp has been determined with TGCE by SpectruMedix's TGCE Systems. However, a preferred size range is from 160 to 800bp.

Screeninq known samples. The TGCE technique can also be applied to genotype known mutation SNPs. To determine all three possible genotypes in a diploid organism (homozygous wild type, homozygous mutant and heterozygous mutant) for a nucleotide alteration at a specific site within an amplicon, a two-pass procedure can be performed. In the first pass, amplicons obtained from each individual are directly analyzed. Any heterozygous individuals will be identified. However, the homozygous wild type and the homozygous mutant are usually not differentiated since they all appear as single peaks. Therefore, a second pass is implemented in which the amplicons from all individuals identified as homozygotes in the first pass are mixed with the amplicon obtained from the wild type. The process will turn a homozygous mutant into a heterozygous mutant upon mixing, while the Status of the homozygous wild type will remain unchanged. Thus, all three genotypes can be determined through these two passes.

The electropherogram or peak pattern generated by TGCE under a Set of fixed conditions is highly reproducible and is related to the characteristics of amplicon, location of the mutation/SNP site and type of mutations (single nucleotide alteration vs. deletion/insertion). The so-called "signature" peak morphology has a significant implication for diagnostic applications where unanticipated mutations may be present and subsequently identified by the different profile. Similarly, the same principle can be applied for mutation/SNP discovery and screening in a highly heterozygous organism where a wild type control with a SNP (already a heterozygote) may be compared with a mutant heterozygote with an additional induced nucleotide alteration.

Overview of System Software and Functions

•     This system is fully automated and controlled by the SpectruMedix CheckMate TM operational software package. The software provides for run method file creation and storage, which allows you to easily create application-specific methods and recall them for future use. Data acquisition parameters are preset and once the system is configured, there aren't any run-to-run or day-to-day adjustments required, other than the occasional replacement of the capillary cartridge.

Additional Features:

•     CheckMate TM provides automated Operation for up to six sample trays.

•     The software has current monitoring capability starting with the Pre-Run stage and continuing through the end of camera Data Acquisition. These measured currents are stored in a tab-delimited text format as a *.curfile, and are useful diagnostic tools for troubleshooting Instrument and/or chemistry issues related to electrophoresis.

•     Temperature control of the cartridge is a feature of CheckMate TM . These data sets are recorded as a *.tmp file, and can be useful for post-run diagnostics.

•     A report file is recorded for each run denoting all relevant running condition information including: voltages, solution information, camera settings, sample information, and the start/stop times for each step of the run. The report file *.rpt can be recalled after the run has finished for review.

Hardware Components

The system includes the following-.

•     Multiplexed Capillary Electrophoresis Instrument — sample loading carousels and elevator, temperature control module, high voltage buffer system, laser, CCD imaging device and electronic controls

•     Instrument CPU — Computer, control cards, Gables, monitor, mouse and keyboard

•     Solution Containers — wash bottles, buffer bottles, gel syringes, and gel bottles

•     Capillary Cartridge — completely self-contained capillary array

•     UPS - uninterruptible power supply

•     Sample and Buffer Tray Holders - Six universal sample tray holders and six buffer tray holders

•     Documentation — Operators manual, software manual, chemistry protocol, and specific application notes

Summary of a Sample Run

A fully automated sample run performs the following operations:

•     capillary preparation — removal of the used gel matrix from the previous run to eliminate memory effects or contamination.

•     Gel matrix injection — the capillary array is filled with new gel matrix.

•     Sample injection — electro kinetic injection of samples.

•     Current monitoring — A detailed history for each capillary can be viewed. A current vs. time plot is created and allows problems to be isolated to a specific cause.

•     Data acquisition — creates a binary *.dmp fite at a rate of approximately 1 MB per minute.

Maintenance Procedures for the Instrument

Pre-Run Check (perform prior using the instrument)

1. Ensure all solution/buffer lines are free of lange air bubbles. Prime lines if necessary.

2. Place a new/full rinse tray on the instrument. Place a new full running buffer tray on the

     instrument for each sample tray you are loading.

3. Check all reagent levels including gel to ensure adequate levels are present for anticipated

     runs. Fill or change if required.

4. Ensure that the lasen cooing fan is running. If the fan is not running, DO NOT operate the

     instrument, and call SpectruMedix Instrument Services immediately.

5. Verify the lasen power comes up to normal levels when CheckMate TM is started. This

     ensures an interlock was not tripped or opened in your absence. If Laser power does not

     come up to normal levels, reset the lasen breaker on the side of the instrument, and wait

     to ensure normal Operation.

6. Make sure that there are no other programs are running on the computer,

7. Verify that there are not any red LEDs flashing on the front instrument panel.

8. Check the walte bottle level and empty if necessary.

Daily Maintenance (even if instrument is not in use)

1. Ensure the rinse level in the rinse tray does not fall below 2/3 full.

2. Reboot the computer.

Weekly Maintenance

1. Remove all data from C:IData. Archive your data using your CD burner or place the data

     on your network.

2. Delete all *.tmp fites from the Windows Temp directory.

3. Defragment your hand drive. Even if Windows indicates that it's not necessary, do it

     anyway.

4. Reboot your computer.

Monthly Maintenance

1. Check all filtern in your reagent bottles. Change if needed.

2. Change the HPLC pump wash solution with a fresh 80:20 mixture of 18 M ohm H20:

     Methanol or Ethanol.

3. Check and clean all interior surfaces of the instrument that may haue developed crystalline

     buildup from either buffer or gel matrix. Clean the drip tray that is located under the drain

     tray.

4. Remove and clean the drain tray using warm tap water.

5. Wipe down the outside of the instrument and Computer with a damp cloth to remove any

     surface dust or contaminates.

6. Air compressor maintenance: If your machine has a Thomas, drain the tank using the

     valve underneath the tank. If your machine has a Jun-Air, check the oil level using the

     indicator an the side of the tank, and add oil if necessary.