Biomolecular Technology - Modulo 2 - Teoria (2008/2009)

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Course code
4S00175
Name of lecturer
Barbara Molesini
Number of ECTS credits allocated
1
Academic sector
BIO/11 - MOLECULAR BIOLOGY
Language of instruction
Italian
Period
1° Sem dal Oct 1, 2008 al Feb 6, 2009.

To show the organization of the course that includes this module, follow this link * Course organization

Lesson timetable

Learning outcomes

This course aimed to study in detail the hybridization between nucleic acids and the PCR that are at the basis of many methods for molecular analysis.

Syllabus

NUCLEIC ACID HYBRIDIZATION
1.INTRODUCTION TO NUCLEIC ACID HYBRIDIZATION: FORCES STABILIZING DNA STRUCTURE, STABILITY OF NUCLEIC ACIDS, EFFECTS OF pH, EFFECTS OF TEMPERATURE, MELTING TEMPERATURE Tm, FACTOR AFECTING Tm
2.TYPES OF HYBRIDIZATIONS
3. SOLUTION HYBRIDIZATION: REASSOCIATION OF DNA, FACTORS AFFECTING REASSOCIATION/HYBRIDIZATION (FACTORS AFFECTING RATE OF HYBRIDS FORMATION AND STABILITY OF HYBRIDS)
4.FILTER HYBRIDIZATION: TYPES AND PROPERTIES OF SUPPORT, TRANSFER OF NUCLEIC ACID TO FILTER, FIXING THE DNA TO THE MEMBRANE, SCREENING OF RECOMBINANT LIBRERIES, DOT/SLOT BLOT AND REVERSE DOT/BLOT, SOUTHERN BLOT, NORTHERN BLOT (RNA EXTRACTION AND GEL ELECTROPHORESIS OF RNA SAMPLES)
5.CHOICE OF PROBES: DNA OR RNA ? RADIOACTIVE AND NONRADIOACTIVE PROBES, DIRECT AND INDIRECT LABELING, LABELING, UNIFORM LABELING OF THE PROBE (NICK-TRANSLATION, RANDOM PRIMED LABELING, PCR-MEDIATED LABELING, RUN-OFF TRANSCRIPTION, CHEMICAL METHODS), END LABELING (T4 POLYNUCLEOTIDE KINASE, FILL-IN BY KLENOW, DNA TAILING BY TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE), DETECTION OF RADIOCTIVE HYBRIDS (AUTORADIOGRAPHY), DETECTION OF NONRADIOCTIVE HYBRIDS (ALKALINE PHOSPHATASE, HORSERRADISH PEROXIDASE, CHROMOGENIC SUBSTRATES, CHEMIOLUMINESCENT SUBSTARTES)
6.SPECIFIC APPLICATIONS OF FILTER HYBRIDIZATION: DNA MAPPING, DETECTION OF RELATED SEQUENCES, DNA FINGERPRINTING BY HYBRIDIZATION, DETECTION OF MUTATIONS, DETECTION OF METHYLATIONS


PCR
1.WHAT IS PCR?
2.REAGENTS: EFFICIENCY, SPECIFICITY, FIDELITY
3.PCR CYCLE. FINAL NUMBER OF COPIES OF THE TARGET SEQUENCE
4) 4.AMPLIFYING THE CORRECT PRODUCT: DETECTION AND ANALYSIS OF PCR PRODUCTS, HOW TO AVOID CONTAMINATION (URACIL N-GLYCOSYLASE, UV, ENZYMATIC TREATMENT), HOT START, NESTED PCR
5.TECHNIQUES AND APPLICATIONS: 5’RACE-PCR AND 3’RACE-PCR, RT-PCR,PCR MUTAGENESIS (DELETION OF SEQUENCES, BASE SUBSTITUTIONS, INSERTION MUTAGENESIS), MODIFICATION OF PCR PRODUCTS(INTRODUCTION OF RESTRICTION SITES, ADDING PROMOTERS AND RIBOSOME-BINDING SITES), JOINING OVERLAPPING PCR PRODUCTS, QUANTITATIVE PCR

Assessment methods and criteria

written examination

Studying