Complement C4 Gene Copy Number Variation Genotyping by High Resolution Melting PCR
Abstract
:1. Introduction
2. Results
2.1. C4 CNV GRACE-PCR Design and Optimization
2.2. Validation of the C4 CNV Determined by HRM-PCR/GRACE-PCR Assay
3. Discussion
4. Materials and Methods
4.1. Samples
4.2. Blood DNA Extraction
4.3. DNA Quantification
4.4. qPCR Oligonucleotides
4.5. Number of PCR Cycles
4.6. Optimization of HRM-PCR/GRACE-PCR Assay
4.7. Standard Curve
4.8. Data Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
C4A | Complement component C4A |
C4B | Complement component C4B |
C4L | long C4 gene |
C4S | short C4 gene |
CNV | Copy Number Variation |
GRACE | Gene Ratio Analysis Copy Enumeration |
HERV-K | Polymorphic endogenous retrovirus insertions |
HRM | High Resolution Melting |
STK19 | serine/threonine kinase 19 (STK19), also known as HLA-RP1, here referred to as RP1 |
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Cell Line. | C4A | C4B | C4S | C4L | Haplotypes |
---|---|---|---|---|---|
COX | 0 | 2 | 2 | 0 | BS BS |
WT51 | 4 | 0 | 0 | 4 | AL-AL AL–AL |
MADURA | 2 | 2 | 4 | 0 | AS-AB AS-BS |
CB6B | 4 | 4 | 2 | 6 | AL-AL-BL-BS AL-AL-BL-BS |
WT8 | 1 | 2 | 0 | 3 | AL-BL BL |
DAUDI | 2 | 1 | 1 | 2 | AL-BS AL |
MANIKA | 2 | 3 | 3 | 2 | AL-BS-BS AL-BS |
HOM2 | 3 | 2 | 1 | 4 | AL-AL-BS AL-BL |
Target | Primers | Primer Sequence (5’ →3’) | Amplicon Size (bp) | Amplicon Tm (°C) |
---|---|---|---|---|
C4A | C4A-F | CCTTTGTGTTGAAGGTCCTGAGTT* | 141 | 84.8 |
C4A-R | TCCTGTCTAACACTGGACAGGGGT* | |||
C4B | C4B-F | TGCAGGAGACATCTAACTGGCTTCT* | 86 | 81.6 |
C4B-R | CATGCTCCTATGTATCACTGGAGAGA* | |||
C4S | C4S-F | TTGCTCGTTCTGCTCATTCCTT* | 103 | 81.5 |
C4S-R | GGCGCAGGCTGCTGTATT* | |||
C4L | C4L-F | TTGCTCGTTCTGCTCATTCCTT* | 133 | 83.7 |
C4L-R | CCAATGGACTTCAGGAACCC | |||
RP1 | RP1-F | GACCAAATGACACAGACCTTTGG* | 79 | 77.6 |
RP1-R | GACTTTGGTTGGTTCCACAAGTC* |
Gene | Final Concentration | Tube |
---|---|---|
C4A | 0.5 µM | Tube 1 |
RP1 | 0.33 µM | |
C4B | 0.21 µM | Tube 2 |
RP1 | 0.16 µM | |
C4S | 0.25 µM | Tube 3 |
RP1 | 0.5 µM | |
C4L | 0.5 µM | Tube 4 |
RP1 | 0.25 µM |
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Jaimes-Bernal, C.P.; Trujillo, M.; Márquez, F.J.; Caruz, A. Complement C4 Gene Copy Number Variation Genotyping by High Resolution Melting PCR. Int. J. Mol. Sci. 2020, 21, 6309. https://doi.org/10.3390/ijms21176309
Jaimes-Bernal CP, Trujillo M, Márquez FJ, Caruz A. Complement C4 Gene Copy Number Variation Genotyping by High Resolution Melting PCR. International Journal of Molecular Sciences. 2020; 21(17):6309. https://doi.org/10.3390/ijms21176309
Chicago/Turabian StyleJaimes-Bernal, Claudia P., Monte Trujillo, Francisco José Márquez, and Antonio Caruz. 2020. "Complement C4 Gene Copy Number Variation Genotyping by High Resolution Melting PCR" International Journal of Molecular Sciences 21, no. 17: 6309. https://doi.org/10.3390/ijms21176309