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>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.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
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