Q-FISH (CAT#: STEM-MB-1178-WXH)

Introduction

Quantitative Fluorescent in situ hybridization (Q-FISH) is a cytogenetic technique based on the traditional FISH methodology. In Q-FISH, the technique uses labeled (Cy3 or FITC) synthetic DNA mimics called peptide nucleic acid (PNA) oligonucleotides to quantify target sequences in chromosomal DNA using fluorescent microscopy and analysis software.

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Principle Applications Procedure Materials Notes Related Products

Principle

Due to the fact that PNA backbones contain no charged phosphate groups, binding between PNA and DNA is stronger than that of DNA/DNA or DNA/RNA duplexes. Q-FISH utilizes this unique characteristic of PNAs where at low ionic strengths, PNAs can anneal to complementary single-stranded DNA sequences while single-stranded DNA cannot. By using conditions that only allow labeled (CCCTAA)3 PNA to hybridize to (TTAGGG)n target sequences, Q-FISH is able to quantify the hybridization of PNAs to telomeric sequences.

Applications

Study telomere length.

Procedure

1. Prepare metaphase-arrested cells
2. Fix cells
3. Prepare slides
4. Fix slides in formaldehyde
5. Treat slides with pepsin
6. Hybridize PNA probe (Cy3 or FITC labeled PNAs)
7. Heat denature DNA
8. Wash slides to remove unbound PNAs and counterstain DNA (DAPI or PI)
9. Image capture and analysis

Materials

• Flow cytometer
• Fluorescence microscopy

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