Internal standard calibration heterozygous detection using high resolution melting data Summary: Background : High-resolution melting curve techniques for genotyping are simple and effective. In a fusion-based approach, the probe method is the gold standard for genotyping, which completely distinguishes the allele from the target. Small fragment genotyping is a genotyping method that replaces probes. The presence of a heteroduplex nucleic acid molecule makes it easy to detect homozygotes with a significant change in the shape of the melting peak. Genotyping of homozygous G/C or A/T is difficult because of the similar Tm values ​​of alleles. The homozygote changes in these base pairs are difficult to distinguish by the small fragment method. Changes in temperature, buffer, and volume between samples limit the separation of homozygotes. Regardless of how accurate the instrument is, changes in user operations need to be noted, and methods that reduce this change can improve small segment genotyping. These limitations can be overcome by using internal specific reaction criteria. background: method: result: Figure 1: Derivative melting curve showing the melting peak of the calibration and amplicon. The left and right peaks are the peaks that calibrate the internal standard. The anatomical profile of 94, near 76 ° C, was generated from an independent PCR reaction. The calibration molecules did not have a clear separation of the homozygous amplification peaks. The narrowest and tallest peak in the middle is the CPS1A/T polymorphic A/A (blue) and T/T (red) homozygous genotypes. The added color is based on previous probe genotyping and is not based on small fragment genotyping data. The wider intermediate peak forms an A/T heterozygote. In this illustration, the top of the magnified peak is displayed. Table 1: Separation of Tm values ​​before and after calibration for each gene of interest from one of the four plates. In all cases, the Tm value becomes lower after calibration. It can be seen from the low standard deviation and non-intersection range after calibration. Table 2: Empirical data shows the effect of correcting sensitivity. Improvements in the nature of sensitivity can be displayed in the calibration data. The wrong name is based on a similar Tm value, not the Lightsanner software term explained in the article. A mispredicted prediction of the amplicon generation for each small target fragment is shown. The software automatically refers to subsequent work, taking into account the development of algorithms within Lightsanner's commercial software, automatically assigning samples to groups of similar shapes. This shape-based genotyping does not require known Tm values. This shape-based designation benefits from correction before a preferred way to confirm the genotype based on the Tm value. Figure 3 shows the results of a plate that improves scanning of the small amplicon product of CPS1. Figure 3: Calibration improves the name of the Lightsanner commercial software automation software. Each screenshot shows a corrected melting curve at the top and a different floor plan at the bottom. In addition, you can see the grouping situation. The red, blue, and gray in the upper left image of the screenshot should be repeated to mimic the comparison of columns 1-6 and 7-12. In this data set, we obtained a sensitivity of 92% before calibration and a sensitivity of 100% after calibration. Calibration reduces the Tm value. The reaction volume depends on the expected Tm value depending on the reaction volume, because more thermal mass in the pores of the 96-well plate contains more mastermix or mineral oil to block heat transfer. This has an effect on increasing the surface Tm value. We used the OTC small amplicon to detect the Tm dependence above the mastermix volume. The volume increased between 7.0-12.5 ul in 0.5 ul increments (10 ul recommended by the instrument manufacturer). The dependence of the Tm value was firm before calibration. (slope=+0.069, R2=0.798), but it is different after correction (slope=-0.001, R2=0.066) (see Figure 4). The effects of different Tm values ​​for different volumes are effectively eliminated by the calibrator. Figure 4: Effect of Mastermix volume change on Tm values ​​before and after calibration. 47 samples were amplified and the change of the OTC gene c.299-8T>A was evaluated. The prepared PCR reaction solution consisted of 1 ul of template DNA, a volume change of Mastermix, from 7.5 to 12.5 ul, with an increase of 0.5 ul per step. Panel A shows the melting before correction, with significant changes. Plate B shows the correction after melting, reducing the variation. Panel C shows the dependence of the Tm value on the Mastermix volume prior to calibration, and the 35 T/T homozygous samples of the reaction were scanned. After the correction, two things can be observed: 1) compress the error bars, indicating a reduction in the change. 2) The slope and R2 values ​​are significantly reduced, showing a small relationship between the Tm value and the Mastermix. The sample corresponding to each reaction volume is distributed somewhere in the plate, eliminating the dependence on the position of the plate. Conclusion Small fragment melting is a simple genotyping method that does not require subsequent PCR operations. Small amplicon is suitable for genotyping for several reasons. However, homozygous genotypes can be successfully distinguished without correction, depending not only on the high-resolution melting data collected, but also on the consistency of buffer, volume, and extraction between samples. The calibrator effectively removes many of the confounding variables. A better, relatively stable measurable, accompanying, better homozygous genotyping is granted. Best Water Distiller,Distilled Water Machine,Podiatry Water Distiller,Portable Water Distiller ZHEJIANG FOMOS MEDICAL TECHNOLOGY CO.,LTD. , https://www.ifomos.com
Method : Synthetic calibrated nucleotides are synthesized at the same concentration as their complementary strands. The PCR reaction was carried out using a conventional PCR machine with the presence of LC Green and an internal standard. After the PCR reaction, it was placed in a LightSanner and scanned for data in the range of 55 °C to 97 °C. Standard melting is characterized by a line that is used to convert the fluorescence data of each sample. The genotype was confirmed after sequencing. We evaluated the error rate of homozygous genotyping before and after correction by measuring the distance between the peak shape of each sample and the average Tm value consistent with the genotype. An error can be observed when the Tm value of the sample is close to the incorrect group average Tm value.
RESULTS : The genotyping sensitivity of base pair SNP mutations increased from 90% to 99% by scanning a large number of sample plates and calibrating the PCR targets. After calibration, the Tm standard deviation was reduced from 0.056 to 0.027 °C.
Conclusion : Temperature internalization within small amplicons can improve genotyping of G/C and T/A base changes. This work was funded by the National Institutes of Health R44DK069106 and R44HD053215 to SFD and R42GM072419 and R42GM073396 to CTW.
Due to the genetic properties of nucleotides, denaturation or melting, the characteristics between genotypes are diverse. Probe-based genotyping has considerable specific allelic temperature shifts - a few degrees Celsius. High resolution provides a detailed map of all double-stranded transitions, making it possible to exclude probe methods. In addition, when the total amplification was detected, the density of the number of bases per experiment was 20 times. Nonetheless, it is difficult for the probe system to detect homozygotes because of potentially similar melting temperatures and melting shapes. The small fragment method provides a simple genotyping solution. 
In particular, small fragment melting provides better homozygous detection because the Tm value shift for a particular genotype is large compared to a large amplicon. However, some factors may prevent the separation of homozygotes. The Tm variability caused by the type of base and instrumental changes and the shift between samples can be genotyped. The calibrator reduces changes in many sources, strains melting and increases sensitivity to genotyping. The best results are obtained when the primers of the small fragments are tightly bound to the sides of the SNP of interest.
The PCR reaction system is 10ul, including 1×Lightsanner high-sensitivity genotyping matermix (Idaho Technology), including hot start enzyme, Mg2+, buffer and other components. The nucleic acid internal standard provides PCR-independent at approximately 62 ° C and 92. The melting curve of °C. Primers include 0.10 or 0.15 um, human genomic DNA 10-15 ng. The PCR reaction conditions were as follows: denaturation at 95 ° C for 2 min, 1 cycle; 94 ° C for 30 s, 63 ° C - 67 ° C (depending on the experiment) 30 s, 45 cycles, combined with annealing and extension. The final annealing step was 28 ° C for 30 s. All DNA samples were analyzed by probe method for genotyping.
Scanning was performed on a LightScanner 96 (Idaho Technology) and data was collected at a temperature ranging from 55 °C to 97 °C. The melting data is close to the fluorescence data after being calibrated by a smooth curve. Second, the Tm value of each internal standard is calculated by the difference vertices of the spline data algebra, and each calibration is arranged by applying the offset and the linear scale factor. This movement process aligns the melting curves of the amplicon so that the minimization becomes smaller. The data was resampled using a cubic spline function curve ruler and analyzed using Lightsanner software. The temperature range around the Tm value of the amplicon (with/without the internal standard calibrated in advance) is analyzed to show the derived peak.
Figure 1 shows the complete melting peak display before calibration. The melting signal is distinguished from the low temperature and high temperature internal standard and the middle of the amplicon melting. 
Figure 2: Derived melting peaks improved with internal standards. The fluorescent signal is shown on the plate and the plate contains 94 independent PCR reactions for changes in CPS1, OTC, MSH2 and PAH. CPS1 bimodal heterozygotes are easily distinguished before calibration (Figure 2a). In contrast, the OTC heterozygote (Fig. 2c) shows only a distinct peak, which is difficult to determine before calibration. Analysis of the internal standard data can easily determine the genotype, and different genotypes can be clearly observed in the replication reaction. 
Table 1: Each amplified plate included the same set of 47 genomic DNA samples (94 independent PCR reactions). The predicted Tm values ​​for all homozygotes are listed on a near-parameter basis. The Tm values ​​of heterozygotes are not included because they are, in general, genotyped in our system without the use of internal standards. The Tm value of the observed value is higher than the theoretical value by 5-7 °C. To some extent, LC Green dyes can make DNA:DNA hybrids more stable. Although the genotype classification without internal standard was rough, the average Tm values ​​after uncalibrated and after calibration were significantly different (p < 0.001, non-parametric, U-test).
The CPS1 and OTC PCR products, adjacent analysis, predicted the same Tm for homozygous T/T and A/A amplicons. However, for the two genes of interest, the average Tms between the two homozygous subgroups is different, even if not corrected. Nevertheless, before correcting the overlap between T/T and A/A homozygotes, as shown in the range of Tm values. After the correction, there was no overlap between T/T and A/A homozygotes, indicating genotype separation. MSH2 and PAH small fragment products, proximity analysis predicted a slight difference in Tm values ​​between amplicon including one of the homozygotes. Analysis of the two changes prior to calibration showed coarse genotyping. After the correction, the type of homozygote is determined. Table 2 shows the incorrect designation of homozygous genotypes. 
*The number of rss that do not specify OTC T/A. 
