CITOGENOMICA NELLA DIAGNOSI PREIMPIANTO E PRENATALE
Academic Year 2022/2023 - Teacher: VINCENZA BARRESIExpected Learning Outcomes
The Molecular Diagnostic Technologies have broad application in the biomedical field and the course aims to give the student the knowledge of
methods and tools useful for the diagnosis of pathologies transmissible to the embryo.
Course Structure
Oral lectures integrated by laboratory activities.
Should teaching be carried out in mixed mode or remotely, it may be necessary to introduce changes with respect to previous statements, in
line with the programme planned and outlined in the syllabus.
Required Prerequisites
Knowledge of chemistry and biochemistry, cellular and molecular biology, genetics, anatomy and physiology acquired during the three-year degree
Attendance of Lessons
Weekly attendance in the first semester, first academic year as required by the Degree Course Regulation.
Detailed Course Content
Biological samples: types, blood and biological samples, processing and storage.
Extraction and purification of nucleic acids from human cells by chromatographic columns for gel filtration, ion exchange and affinity.
Purification of nuclec acids with magnetic beads.
Quantitative assay for nucleic acids using spectrophotometric and fluorimetric methods.
Technologies based on the complementarity of the purine and pyrimidine nucleotides.
Synthesis of cDNA by reverse transcription.
Polymerase chain reaction (PCR). Qualitative PCR or End Point and quantitative PCR or Q-Real Time PCR. Programs for the design of
primers to be used for PCR reactions.
Evaluation of gene expression in real time by the method of 2-ΔΔCT. for Allelic discriminationusing the Real Time PCR. Restriction and
Modification enzymes. Agarose gel electrophoresis for the separation of nucleic acids. Capillary electrophoresis for acid nucleic sequencing and analysis of
microsatellite.
Protein extraction and analysis. Electrophoresis of proteins. Western blot.
DNA- and RNA-microarrays: Preparation of samples of DNA or RNA for the global analysis of the genome and transcriptome. Molecular
analysis of the human karyotype microarray genomic photolithography-based probes to "Single Nucleotide Polymorphism-SNP" and "Copy
Number Variation-CNV". Using algorithms and softwares for the interpretation of the data (CN state, Allele difference). Transcriptome analysis.
Microarray expression to oligonucleotides obtained by photolithography synthesis. Array-CGH. Using algorithms and softwares for data
interpretation (RMA and SAM).
Application of the "microarray" technology for the study of solid and hematological tumors.
Acid Nucleic First-generation sequencing technology, second generation and third generation. Methods for the preparation of libraries for the
exome analysis and specific regions using a multiplex. Methods of clonal amplification. Techniques for the detection of incorporated
nucleotides. Bioinformatics and statistical methods for interpreting data obtained from second-generation sequencing platform (NGS).
Implementation of the first generation sequencing techniques for the detection of mutations with prognostic and predictive significance of
response to therapy. Application of second generation technologies for the analysis of solid and hematological tumors.
Detection of chromosomal aberrations by MLPA technology (Multiplex Ligation-dependent Probe Amplification).
Human cell cultures.
Textbook Information
1. Additional material supplied by the teacher; 2) Fundamental Molecular Biology; Lizabeth A. Allison, Editore: John Wiley & Sons Inc; 3)
Online courses: http://www.dsf.unict.it/index.php?page=progetto-phar-in; "Riparazione del Dna e mutagenesi nel cancro" (D.F.
Condorelli); Mismatch repair (MMR) instabilità dei microsatelliti (V. Barresi); "Tecnologie avanzate per la Bio-Medicina Genomica"
(Vincenza Barresi).
Author | Title | Publisher | Year | ISBN |
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Course Planning
Subjects | Text References | |
---|---|---|
1 | Biological samples: types, sampling, treatment and storage. | 1) Metodologie Biochimiche e Biomolecolari, a cura di Maccarone M. e colleghi, Zanichelli editore. 2) Scientific papers and teaching material provided by the teacher of the course |
2 | Extraction and purification of nucleic acids from human cells by means of chromatography columns for filtration gel, ion exchange and for affinity. Purification of nucleic acids with magnetic beads. Dosage and qualitative evaluation of nucleic acids with spectrophotometric and fluorimetric methods. | 1) Metodologie Biochimiche e Biomolecolari, a cura di Maccarone M. e colleghi, Zanichelli editore. 2) Scientific papers and teaching material provided by the teacher of the course |
3 | Technologies based on the complementarity of purine and pyrimidine nucleotides. | 1) Metodologie Biochimiche e Biomolecolari, a cura di Maccarone M. e colleghi, Zanichelli editore. 2) Scientific papers and teaching material provided by the teacher of the course |
4 | Synthesis of cDNA by reverse transcription. Polymerase chain reaction (PCR). Various types of PCR. Restriction and modification enzymes | 1) Metodologie Biochimiche e Biomolecolari, a cura di Maccarone M. e colleghi, Zanichelli editore. 2) Scientific papers and teaching material provided by the teacher of the course |
5 | Agarose gel electrophoresis of nucleic acids. Capillary electrophoresis of nucleic acids for the analysis of single nucleotides and for the analysis of microsatellites. | 1) Metodologie Biochimiche e Biomolecolari, a cura di Maccarone M. e colleghi, Zanichelli editore. 2) Scientific papers and teaching material provided by the teacher of the course. |
6 | First generation, second generation and third generation nucleic acid sequencing technologies. Methods for the preparation of libraries for the analysis of the exome and specific regions via multiplex. Clonal amplification methods. Techniques for the detection of incorporated nucleotides. Bio-informatic and statistical methods for the interpretation of data obtained from second sequencing platforms generation (NGS). Applications for the study of pathologies transmissible to the embryo. | 1) Metodologie Biochimiche e Biomolecolari, a cura di Maccarone M. e colleghi, Zanichelli editore. 2) Scientific papers and teaching material provided by the teacher of the course. |
7 | DNA- and RNA-microarrays: Preparation of DNA or RNA samples for global genome and transcriptome analysis. Molecular analysis of the human karyotype by means of photolithographic genomic microarrays based on “Single Nucleotide Polymorphism-SNP” and “Copy Number Variation-CNV” probes. Use of algorithms and softwares for data interpretation (CN state, Allele difference). Transcriptome analysis by expression microarray. CDNA microarrays. Oligonucleotide expression microarrays. Applications for the study of pathologies transmissible to the embryo. | 1) Metodologie Biochimiche e Biomolecolari, a cura di Maccarone M. e colleghi, Zanichelli editore. 2) Scientific papers and teaching material provided by the teacher of the course |
8 | Detection of chromosomal aberrations using MLPA (Multiplex Ligation-dependent Probe Amplification) technology. | 1) Scientific papers and teaching material provided by the teacher of the course. |
Learning Assessment
Learning Assessment Procedures
Oral exam. Learning assessment may also be carried out on line, should the conditions require it.
Examples of frequently asked questions and / or exercises
Applications of first generation sequencing. Microarrays analysis applications to preimplantation diagnosis for the evaluation of embryonic chromosomal aneuploidies. Parameters for the detection of CN-LOH. Application of "NGS" sequencing for RNA analysis.
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