American Society of Cytopathology Core Curriculum in Molecular Biology

Transcription

1 American Society of Cytopathology Core Curriculum in Molecular Biology

2 American Society of Cytopathology Core Curriculum in Molecular Biology Chapter 3 Molecular Techniques Fluorescence In Situ Hybridization Keisha N. Brooks, MS, CT, MB(ASCP) University of Tennessee Health Science Center Memphis, Tennessee

3 Learning Objectives Define FISH and its purpose for use in the clinical laboratory Distinguish between FISH and CISH Explain the basic steps in an ISH procedure Identify the cause of common problems in the procedure Describe amplification techniques used to detect small targets

4 In situ hubridization (ISH): Purpose A technique for detecting and localizing nucleic acid sequences in chromosomes, cells, and tissue segments Allows detection of specific nucleic acid sequence targets inside individual cells while preserving cell and tissue morphology Allows for the simultaneous assessment of morphologic alterations associated with genetic composition of the cells Identification and characterization of numerical and structural chromosome abnormalities. Detection of microscopically invisible deletions. Detection of sub telomeric aberrations. Prenatal diagnosis of the common aneuploidies (interphase FISH).

5 Fluorescent in situ Hybridization (FISH) Hybridization of complementary gene or regionspecific fluorescent probes to chromosomes. Interphase or metaphase cells on slide (in situ) Probe Microscopic signal (interphase)

6 Sample/Target in situ means in place (hybridization takes place in the cell where the target DNA or RNA is normally present) Samples: Frozen or paraffin embedded tissue Cytology preparations: body fluids, FNA s hematologic specimens, etc Cytogenetic preparations (metaphase preparation) Cultured cells Interphase nuclei *Fixed specimens (e.g., formalin fixed) usually require pre treatment for target access

7 Fluorescent in situ Hybridization (FISH) Metaphase FISH Chromosome painting Spectral karyotyping Interphase FISH

8 in situ hybridization (ISH) ISH FISH Fluorescent labeled probes Viewed with fluorescent microscope CISH Chromogenic detection Viewed under light microscope

9 Types of Probes DNA Requires denaturation before hybridizing Available in short oligonucleotide probes Good target penetration More stable that larger megabase DNA probes RNA ( riboprobes ) Single stranded (no initial denaturation needed) Synthesized from a vector via an RNA polymerase Hybridize better to DNA targets but unstable die to ubiquitous presence of RNAses PNA (peptide nucleic acids) Same bases as DNA or RNA probes but joined by the backbone of amide linkages (like proteins) instead of sugar and phosphate in DNA/RNA backbone Kinetic properties are different and not yet well understood Hybridize more rapidly with DNA probes and with better homology

10 FISH Probes Chromosome specific centromere probes (CEP ) Hybridize to centromere region Detect aneuploidy in interphase and metaphase Chromosome painting probes (WCP) Hybridize to whole chromosomes or regions Characterize chromosomal structural changes in metaphase cells Unique DNA sequence probes (LSI ) Hybridize to unique DNA sequences Detect gene rearrangements, deletions, and amplifications

11 FISH Probes Telomere specific probes (TEL) Hybridize to subtelomeric regions Detect subtelomeric deletions and rearrangements Probe binding site Telomere Unique sequences kb 3 20 kb Telomere associated repeats (TTAGGG)n

12 Genetic Abnormalities by Interphase FISH LSI Probe Normal diploid signal Brought Greater or less than two signals per nucleus is considered abnormal. Cell nucleus Trisomy or insertion Monosomy or deletion

13 Structural Abnormality by Interphase FISH LSI Probe (Fusion Probe) Brought

14 Structural Abnormality by Interphase FISH LSI Probe (Break Apart Probe)

15 Translocation by Metaphase FISH WCP Probe (Whole-Chromosome Painting) Brought

16 Summary Fluorescence in situ hybridization (FISH) is a popular method used to detect protein, RNA and DNA structures in the cell or in situ The use of FISH is beneficial in detecting subtle chromosomal changes during interphase and metaphase of the cell cycle The principle difference between FISH and CISH is in the types of probes used in analysis. FISH uses fluorescent probes, and CISH uses chromogenic probes.