Steps
Step 1
Genetic Disease Selection and Molecular Genetics Study Design
The core task of this step is to select a genetic disease and design a molecular genetics study plan. Choose from monogenic disorders, polygenic disorders, chromosomal disorders.
• Select disease for study: choose from Mendelian disorders (thalassemia, hemophilia, PKU, Huntington disease, Duchenne muscular dystrophy), complex diseases (diabetes, hypertension, schizophrenia), chromosomal disorders (Down syndrome)
• Study design: determine study type—disease gene mapping and cloning (family linkage analysis, candidate gene approach, WES, WGS), genotype-phenotype correlation study, disease mechanism study (cell/animal models), molecular diagnostic method development, population genetics study
• Sample collection plan: determine sample size and types (patients, family members, normal controls); develop sample collection criteria (diagnostic criteria, inclusion/exclusion criteria); design clinical data collection form (medical history, signs, test results, pedigree); consider ethical review and informed consent
Deliverable: Molecular genetics study design proposal (disease selection rationale, study design, sample collection plan, technical roadmap) | Quality standard: Research-valuable disease selection, scientific and reasonable design, clear and feasible technical roadmap
Step 2
Sample Preparation and DNA/RNA Extraction
The core task of this step is to prepare study samples and extract high-quality DNA and RNA. Molecular genetics experiments require high nucleic acid quality; high-quality nucleic acids are the foundation of successful downstream experiments.
• Sample collection and processing: collect peripheral blood (EDTA anticoagulant), tissue samples (liquid nitrogen frozen or FFPE), buccal swabs; record sample information (ID, source, collection time, storage conditions); establish sample bank and tracking system
• Nucleic acid extraction and purification: use commercial kits (Qiagen, Thermo Fisher) or traditional methods (phenol-chloroform, salting out) for genomic DNA extraction; use TRIzol or column-based methods for total RNA extraction; work in clean bench to prevent RNase/DNase contamination
• Nucleic acid quality testing: use Nanodrop to measure concentration and purity (A260/A280 ratio, DNA≈1.8, RNA≈2.0); use agarose gel electrophoresis to check DNA integrity (genomic DNA should be single band, no degradation); use Agilent Bioanalyzer for RNA integrity (RIN≥7)
Deliverable: High-quality nucleic acid samples (DNA/RNA samples, concentration/purity test report, integrity assessment results), sample bank records | Quality standard: Nucleic acid concentration and purity meet standards, good integrity, no degradation or contamination, complete sample records
Step 3
Gene Mutation Detection and Analysis
The core task of this step is to detect gene mutations using molecular biology techniques and perform genetic analysis. Select appropriate mutation detection methods based on research purpose and disease type.
• Candidate gene screening: use PCR-Sanger sequencing for candidate gene exon amplification and sequencing—design primers (Primer3, NCBI Primer-BLAST), gradient PCR optimization, agarose gel electrophoresis validation, Sanger sequencing (ABI 3730); use SeqMan or Sequencher to align with reference sequence and identify mutations
• High-throughput sequencing analysis: use whole exome sequencing (WES) or targeted panel sequencing—prepare sequencing libraries (NimbleGen, Agilent capture system); sequence on Illumina platform; bioinformatics analysis (BWA alignment, GATK variant calling, ANNOVAR annotation); filter pathogenic mutations (allele frequency, functional prediction, co-segregation analysis)
• Large fragment variation detection: use MLPA (Multiplex Ligation-dependent Probe Amplification) for exon deletion/duplication detection; use aCGH or SNP array for CNV detection; use FISH for chromosomal translocation or large deletion detection
Deliverable: Gene mutation detection results (sequencing traces, variant list, mutation validation results, genotype data table) | Quality standard: Accurate mutation detection method, comprehensive variant annotation, reliable validation results
Step 4
Mutation Functional Validation and Molecular Mechanism Study
The core task of this step is to validate pathogenicity of mutations and study molecular pathogenic mechanisms. Discovering mutations is only the first step; functional experiments are needed to prove pathogenicity.
• Bioinformatics prediction and co-segregation analysis: use SIFT, PolyPhen-2, MutationTaster, CADD to predict pathogenicity of missense mutations; perform family co-segregation analysis; query HGMD, ClinVar, gnomAD databases for mutation frequency and clinical significance
• Cell-level functional validation: construct wild-type and mutant expression vectors (site-directed mutagenesis PCR); transfect cell lines (HEK293T, HeLa or relevant cells); test—mRNA expression (qPCR), protein expression (Western blot), subcellular localization (immunofluorescence confocal microscopy), protein stability (CHX chase assay), enzyme activity, apoptosis, cell proliferation, etc.
• Animal models and molecular mechanisms: if necessary, construct model organism models (mouse, zebrafish, Drosophila)—CRISPR/Cas9 knockout or knock-in; observe phenotypic changes; perform histopathological examination; explore molecular mechanisms (abnormal signaling pathways, altered protein interactions, transcriptional regulation abnormalities)
Deliverable: Functional study results (bioinformatics prediction, cellular functional experiment results, animal model phenotypes, mechanism analysis) | Quality standard: Rigorous functional experiment design, reliable results, reasonable and in-depth mechanism explanation
Step 5
Molecular Genetics Research Report and Diagnostic Application Development
The core task of this step is to write a molecular genetics research report and develop molecular diagnostic applications. Systematically summarize research findings and clarify molecular genetic basis of the disease.
• Write research report: compose complete report following molecular genetics research standards, including introduction, materials and methods, experimental results, analysis and discussion, conclusions, references; systematically present mutation discovery, functional validation, and mechanism study results
• Molecular diagnostic method development: based on discovered pathogenic mutations, develop molecular diagnostic methods—PCR-RFLP, ARMS-PCR, real-time qPCR, HRM, Sanger sequencing, gene chip, NGS panel; perform methodological validation (sensitivity, specificity, reproducibility)
• Genetic counseling and clinical application: write genetic counseling manual explaining inheritance pattern, recurrence risk, diagnostic methods, prevention measures; develop carrier screening protocol, prenatal diagnosis protocol, PGD protocol; draw pedigree charts
Deliverable: Molecular genetics research report, molecular diagnostic methodology materials, genetic counseling manual | Quality standard: Academic standard research report, feasible diagnostic methods, professional and comprehensive genetic counseling content