ABSTRACT
DNA is the most accessible biologic material for obtaining information from the human genome because of its molecular stability and its presence in every nucleated cell. Currently, single nucleotide polymorphism genotyping and DNA methylation are the main DNA-based approaches to deriving genomic and epigenomic disease biomarkers. Upon the discontinuation of the Schleicher & Schuell IsoCode product (Dassel, Germany), which was a treated paper system to elute DNA from several biologic sources for polymerase chain reaction (PCR) analysis, a high-yielding DNA elution method was imperative. We describe here an improved procedure of the not fully validated Whatman pH-based elution protocol. Our DNA elution procedure from buccal cells collected in Whatman FTA cards (Whatman Inc., Florham Park, NJ) yielded approximately 4 microg of DNA from a 6-mm FTA card punch and was successfully applied for HLA-DQB1 genotyping. The genotypes showed complete concordance with data obtained from blood of the same subjects. The achieved high DNA yield from buccal cells suggests a potential cost-effective tool for genomic and epigenomic disease biomarkers development.
Subject(s)
Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , DNA/isolation & purification , Hydrogen-Ion Concentration , Mouth Mucosa/cytology , Alleles , DNA/blood , HLA-DQ Antigens/genetics , HLA-DQ beta-Chains , Humans , Membrane Glycoproteins/genetics , Statistics as TopicABSTRACT
OBJECTIVES: To provide an update on the molecular procedures used increasingly in the study and diagnosis of a variety of dermatologic malignancies and inflammatory disorders and to explore the potential use of these techniques in clinical dermatology. Herein, we review assays such as G-banding, fluorescence in situ hybridization, comparative genomic hybridization, and spectral karyotyping in conjunction with the polymerase chain reaction and DNA microarrays. DATA SOURCES: PubMed was searched for published articles on molecular diagnosis and dermatologic diseases. STUDY SELECTION: All English-language studies were selected if they provided useful methodologic information or highlighted the usefulness of molecular techniques. DATA EXTRACTION: Only methodologic and qualitative information was extracted. DATA SYNTHESIS: The information was synthesized into 2 sections: one describing the principles of different molecular diagnostic techniques, and the other highlighting the contributions of molecular diagnostic techniques to the understanding and diagnosis of several dermatologic diseases. CONCLUSIONS: A basic understanding of the principles of molecular diagnostic techniques is crucial for the practicing dermatologist to benefit from the increasing number of molecular diagnostic articles appearing in the literature and potentially to apply these methods in clinical practice.
Subject(s)
Molecular Diagnostic Techniques/methods , Skin Diseases/diagnosis , Skin Diseases/genetics , Cytogenetic Analysis , Female , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Male , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Skin Neoplasms/diagnosis , Skin Neoplasms/geneticsABSTRACT
BACKGROUND: Cervical cancer represents a major health problem in Venezuela as well as in other Latin American countries. High-risk human papillomavirus (HR-HPV) infection is known as the major risk factor of cervical cancer. However, whether or not a HR-HPV-infected woman progresses to cervical cancer may depend on the immune system effectors induced by viral antigens presented by her specific human leukocyte antigens (HLA) alleles. The role of the HLA system in presenting peptides to antigen-specific T-cells may be critical for genetic susceptibility and genetic resistance to cervical carcinoma. OBJECTIVE: We aimed to investigate the relationship between HLA-DQB1, HPV infection, and cervical cancer in Venezuelan women. METHODS: Blood samples and cervical swabs were obtained from 36 patients and 79 healthy controls; additional cervical biopsies were obtained from all the patients. HPV DNA was detected by PCR and HLA-DQB1 genotyping was performed using a PCR-SSP protocol. RESULTS.: A positive association with cervical cancer was observed for HLA-DQB1*0201-0202 and *0402 alleles, however after Bonferroni correction only HLA-DQB1*0402 remained statistically significant (P value = 0.004, RR = 5.067). CONCLUSION: This is the first report of HLA-DQB1 alleles associated with cervical carcinoma in Venezuelan women. Larger studies are needed to assess whether these HLA-DQB1*0201-0202 and *0402 alleles have a direct effect on disease susceptibility.
