Figure 14.2 Sample processing steps using ABI310 Genetic Analyzer.
denatured to separate the two strands of each PCR product and then loaded in the instrument for analysis. A typical sample preparation method for the ABI 310 is as follows:
1. To a 0.5 mL tube, add 25 |lL of deionized formamide.
2. Add 1 |lL internal lane standard (GS500-ROX or ILS600).
3. Add 1 |lL PCR product amplified with AmpFlSTR kit or Promega PowerPlex 16.
4. Place grey septum on top of 0.5 mL tube.
5. Heat denature sample for 2-3 minutes at 95°C.
6. Snap-cool the sample on ice for 2-3 minutes.
7. Place the sample in the 48-position autosampler tray (96-position trays also exist).
8. Place the autosampler tray in the ABI 310 instrument.
9. Enter the sample names into the ABI 310 sample sheet.
To simplify the sample preparation process and remove step 2, an equivalent amount of labeled internal lane standard may be added to a batch of deionized formamide. For example, if 50 samples were being prepared at a time, then 1250 ||L of deionized formamide and 50 ||L of GS500-ROX could be combined and 26 ||L aliquoted to each sample tube. Regardless of whether the formamide and ROX-labeled internal standard are added separately or together, each sample tube contains ~27 ||L most of which is deionized formamide, and the PCR amplified sample has been diluted approximately 1:27 in the formamide. This dilution does two things. First, the high concentration of formamide helps keep the DNA stands denatured, especially after they are coaxed apart by heating to 95°C. Second, by diluting the PCR sample the salts are also diluted which aids in the sample injection process.
For the same reason, it is important that the formamide be deionized. A good method for deionizing the formamide is the addition of an Amberlite ion exchange resin to the formamide. It is also a good idea to prepare a large batch of formamide and then aliquot it into single use portions. Subjecting the for-mamide to freeze-thaw cycles can cause it to break down and form ionic byproducts that impact the injection process.
The salt content of a sample is very important in the CE electrokinetic injection process as will be discussed later. Contaminating salts can come from either the formamide or the sample if it is not diluted enough. It should also be noted that deionized water can be used in the place of deionized formamide with the only caveat that samples may not be as stable after several days in water compared to formamide. A description of a procedure involving water instead of formamide for ABI 310 sample preparation has been published (Biega and Duceman 1999).
On the ABI 310 Genetic Analyzer, DNA samples are loaded into the capillary with electrokinetic injection. Each sample is placed in an analysis tube and then a voltage is applied to the sample to help draw it into the capillary opening. Electrokinetic injections selectively introduce a sample's charged species into the capillary. More sample material can be introduced into the capillary by simply increasing the voltage or the time applied. In order for this type of injection to work properly, the capillary and the electrode must both extend deep enough into the sample tube to fully interact with the solution in order to establish current flow during the application of the injection voltage. The standard injection on the default STR typing module is 15 000 V for five seconds.
Signal intensity may be increased by lengthening the sample injection time (e.g., from 5 to 10 or 15 seconds), dialyzing the sample on a filter membrane to remove salt from the solution (McCord et al. 1993a), or suspending the sample in deionized water (Butler 1995). The peaks in a sample's fluorescent signal should be kept between 150 and 6000 relative fluorescence units (RFUs) on the ABI 310 for optimal results. If the peaks are off-scale (above ~7500 RFUs), then the sample can be simply re-injected for a lower amount of time, such as two seconds instead of the standard injection of five seconds, to bring the peaks back on scale.
Electrokinetic injections of DNA samples are highly dependent upon the levels of salt in the samples. These salt levels may be measured with a conductivity meter in terms of microsiemens (||S). The same sample with an identical amount of DNA that is diluted in formamide solutions with different conductivity results in vastly different sensitivity levels (Figure 14.3). As the salt level increases and the sample conductivity goes up, fewer DNA molecules are injected because they are competing with the salt ions to get onto the capillary. In fact, the amount of DNA injected is inversely proportional to the ionic strength of the sample (Butler et al. 2004). This differential sample injection due to salt content of the sample is from a process known as sample stacking.
Data Collection Scan Number w 0 iüULJ_Lü_L
1800 la 900
1800 900 0
2100 2400 2700 3000 3300 3600 3900 4200 4500 4800 5100 5400 5700
Figure 14.3 Results from ABI 310 using the same sample that has been diluted in different formamide solutions. Formamide solutions with higher conductivities (larger number of result in less DNA being injected into the capillary. The sample is the GS350 ROX-labeled sizing standard. Figure courtesy of Bruce McCord, Ohio University.
Sample stacking is the process that results when samples are injected from a solution that has a lower ionic strength than the buffer inside the capillary. The buffer for example may be 100 mM in salts while the sample is less than 1 mM in ionic strength. When the electric field is applied during an electrokinetic injection, the resistance and field strength in the sample plug region increase because there are fewer ions to carry the current in the lower ionic strength sample. This causes the ions from the sample to migrate rapidly onto the capillary. As these sample ions enter a region where the polymer solution and buffer are at higher ionic strength, they stop moving as quickly and stack as a sharp band at the boundary between the sample plug and the electrophoresis buffer (Butler 1995).
One way to think of this sample stacking process is to imagine a flowing stream of water. When the banks are closer together, the water runs more rapidly than when the banks of the stream are further apart. By reducing the ionic strength of the DNA sample, the 'banks of the stream' are brought closer together and the sample rushes more quickly into the capillary. The amount of DNA that loads into the capillary during the period of time that voltage is applied during an electrokinetic injection is thus a function of the sample's ionic strength.
Hence, samples prepared and diluted in water or formamide with a lower conductivity will give the highest degree of stacking. Formamide conductivity has a dramatic influence on the amount of DNA injected onto the capillary and therefore the sensitivity of the STR typing assay. In addition, the quality of the formamide has been shown to impact the resolution of closely spaced alleles such as the TH01 9.3 and 10 alleles that are 1 bp apart (Buel et al. 1998).
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