Prediction method for prostate cancer postoperative GS upgrading
A prediction method, a technology for prostate cancer, applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of rapid disease progression, high BCR of patients, affecting accurate assessment of patients' preoperative conditions and selection of treatment options, etc. To achieve the effect of avoiding misjudgment of the disease
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Embodiment 1
[0051] The present invention provides a prediction method for GS upgrade after prostate cancer operation, and its specific prediction method steps include:
[0052] Step 1, extracting clinical tumor DNA samples, and optimizing the processing of extracted clinical tumor DNA samples;
[0053] Step 2, perform pyrosequencing template PCR amplification, amplify PCR pyrosequencing, design methylation detection PCR primers and sequencing primers, and mark the sequencing primers as S (Table 1).
[0054] After PCR, use the sample pretreatment device provided by Pyrosequencing to prepare single-stranded DNA templates with 20-25 μl of biotin-linked PCR products and streptavidin Sepharose HP beads (Amersham Biosciences, Uppsala, Sweden). Then, 15 pmol of sequencing primers were added to each sample, and the prepared samples were placed on the PSQ 96MA instrument, and detected using the SNP Reagent Kit (Pyrosequencing AB).
[0055] Step 3, carry out pyrosequencing, mix and incubate the PC...
Embodiment 2
[0090] Establishment of CRMP4 methylation detection method:
[0091] The CRMP4 promoter CpG island was predicted according to the base sequence of the CRMP4 promoter (such as figure 2 ) shown. All potential sites of CRMP4 promoter CpG island were marked and methylation detection specific primers were designed (as shown in Table 1).
[0092] CRMP4 gene promoter analysis showed two CpG islands, and BSP sequencing results showed that there were two consecutive methylated regions in metastatic PCa and its lymph node CpG island 1: Region A (methylation sites -848, -841) and Region B (methylation sites -690, -680, -678, -674, -671, -665, -660, -658); and these 10 sites in localized PCa, benign prostatic hyperplasia and normal prostate tissue Spots are unmethylated or have sporadic methylation.
[0093] A: There are two CpG island sequencing peaks in the promoter region of CRMP4, and TSS is the transcription initiation site. After sulfuration treatment and parallel PCR amplifica...
Embodiment 3
[0099] Enter, for example, "CRMP4", and then conduct a gene sequence search. At this time, many sequences of different species will be found. Usually, the samples we make are human, so just select the one with human markers.
[0100] It can be seen that the gene sequence of CRMP4 is reversed. Pull down to see the following information, usually select the first Genomic (compared with recognized sequences), you can view the details of this sequence. Generally, the methylation is in the promoter region (or the first exon region) of the gene, so we need to find the promoter region before this sequence. Usually a length of 1000bp is added before the gene sequence. At this time, you can modify the start and end positions displayed in the gray box. Usually the forward sequence can directly reduce the number by 1000, but we know that this sequence is reversed, so we need to change the number after "to" and add 1000 to update.
[0101] Check the genome sequence: Sometimes there are ...
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