2024年4月10日发(作者：被忽悠办理了两年合约制套餐)

Protocol of PNA Telomere probe for FISH

(Fluorescence in situ hybridization)

Introduction

Telomeres have important functions in the stability and replication of chromosomes. These

functions are mediated by highly conserved repeats which consist of (TTAGGG)n in all

vertebrates. The number of telomeric repeats in human somatic cells appears to decrease with

cell divisions and with age. Telomere shortening may act as a mitotic clock in normal somatic

cells and high levels of the enzyme telomerase (capable of elongating telomeres) have been found

in tumor cells. The most commonly used tool to estimate telomere length is southern analysis of

genomic DNA digested with selected restriction enzymes. Such analysis requires thousands of

cells and provides only a crude estimate of the average number of TTAGGG repeats in the

chromosomes of all cells analyzed. In principle, fluorescence in situ hybridization (FISH) should

be able to provide information on the telomere length of individual chromosomes. Directly

labeled oligonucleotide probes are attractive probes for such analysis because of their small size

(good penetration properties), single strand nature (no denaturation of probe) and controlled

synthesis. However the efficiency of oligonucleotide hybridizations for telomeric repeats has not

been sufficient to extend this approach beyond qualitative studies of TTAGGG repeat sequences

in chromosomes of various species. Recently, it was shown that peptide nucleic acid (PNA)

oligonucleotide probes will hybridize with complementary oligonucleotide sequences and that

the resulting duplexes are more stable than DNA/DNA or DNA/RNA duplexes. In PNA, the

charged phosphate-(deoxy) ribose backbone of conventional DNA and RNA oligonucleotides is

replaced by an uncharged backbone of repeating N-(2-amino ethyl)-glycine units linked by

peptide bonds. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the

higher sequence specificity, improved stability, reproducibility, and lower background. Due to

the higher Tm of PNA/DNA duplexes, short (18-mer) Telomere PNA (CCCTAA)

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are widely used.

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Things to do before starting

I. Prepare the Telomere PNA

1. Centrifuge the tubes before opening them in order to collect lyophilized PNA at the bottom of

the tubes.

2. Add distilled water to each tube to obtain an approximately 67 ㎍/㎖stock solution of each

PNA.

PNA

5 nmol

10 nmol

Distilled water

0.5 ㎖

1 ㎖

Final concentration

67 ㎍/㎖

67 ㎍/㎖

3. Dilute telomere PNA probe in PNA hybridization buffer to a final concentration of 800 ng/㎖

4. Place 15 ㎕of diluted PNA probe onto the specimen.

9Note: Store the Telomere PNA probe solution in the dark at 4 ℃.

II. Prepare Solutions

1. Hybridization buffer

10 mMNaHPO

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, pH 7.4

10 mMNaCl

20 mM Tris, pH 7.5

70% formamide

2. Fixative solution

Prepare Methanol : Glacial acetic acid (3:1) solution.

9Note: Prepare just before use!

3. Pepsin 0.005% solution

2.5 ㎖of 10% Pepsin stock in 50 ㎖0.01 M HCl.

9Note: Make fresh! Warm to 45 ℃before use.

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