Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

Summary

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

Abstract

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern^1,2. Here, we present a detailed description of this procedure, with some modifications.

Introduction

Telomeres, situated at the ends of chromosomes, are nucleotide repeats of the sequence TTAGGG (on human chromosomes) that play a critical role in protecting cellular genetic information^3,4,5. Telomeres are several thousand base pairs in length and serve to protect the integrity of the rest of the chromosome during DNA replication. Replication of chromosomes is not perfect resulting in the ends of the chromosome not being completely copied. This inefficiency in replication is termed the "end-replication problem" and results in loss of some of the telomere repeats durin....

Protocol

1. Genomic DNA Extraction

1. Perform a genomic DNA extraction from the cells (here, cell line BJ-hTERT) to be analyzed using a commercial DNA extraction kit, according to the manufacturer's instructions.
2. Measure the DNA concentration with a spectrophotometer. If the DNA concentration is less than 100 µg/mL, precipitate the DNA with ethanol and resuspend in a volume of TE buffer (10 mM Tris-Cl; 0.1 mM EDTA (Ethylenediaminetetraacetic Acid); pH 8.0), to ensure a DNA concentration of 100-600 µg/mL).

2. DNA Integrity Assessment

NOTE: This portion of the protocol outlines a DNA in....

Results

Three concentrations of DNA (1, 2 and 3 µg) isolated from the cell line BJ-hTERT (telomerase immortalized human foreskin fibroblasts)²² and human peripheral blood mononuclear cells (PBMCs) were analyzed for telomere length. Table 1 shows the lane assignments for each DNA sample. Figure 1 shows a nucleic acid fluorescent stain of the gel. The molecular weight standards are clearly visible. The distance from the top of t.......

Discussion

Following electrophoresis, the genomic DNA smear is higher than 800 bp. This can occur if the restriction enzymes are faulty or if additional enzymes (as described above) are needed. A second possible explanation is that the protein is still attached to the DNA. When determining the DNA concentration by spectrophotometer, the 260/280 ratio should be around 1.8. If this ratio is <1.5, there is protein contamination which can interfere with restriction enzyme digestion. Either the DNA sample would need to be re-isolate.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biologics
Rsa1, restriction enzyme	New England Biolabs, Inc	R0167S	10,000 U/mL, DNA digestion.  Comes with Cutsmart Buffer (digestion buffer)
Hinf1, restriction enzyme	New England Biolabs, Inc	R0155S	10,000 U/mL, DNA digestion.  Comes with Cutsmart Buffer (digestion buffer)
Seakem GTG Agarose	Lonza	50071	electrophoresis grade agarose
Optikinase	affymetrix	78334X 500 UN	T4 Polynucleotide Kinase
SYBR Green Nucleic Acid Stain I	ThermoFisher Scientific	S7567	Syber Green
exactGene DNA Ladder 24- kB	Fisher Scientific	BP2580100
C-Strand Probe (3'-(G3AT2)4-'5)	Integrated DNA Technologies		Custom ordered DNA oligonucleotide
Gamma-ATP-P32	Perkin Elmer	BLU502Z	Radioisotope
Qiagen Blood and Cell Culture DNA Mini Kit	Qiagen	13323	DNA extraction kit
GE Illustra Microspin G-25 column	GE Healthcare Life Sciences	27-5325-01	For purification of readioactive oligo probe
Ficoll 400			non-ionic synthetic polymer
Equipment/Software
Owl A5 Gel electrophoresis system (20 cm x 25 cm)	ThermoFisher Scientific	A5	Gel electrophoresis
Horizon 11.4 Gel electrophoresis system (11 cm x 14 cm)	Corel Life Sciences	11068020	Gel electrophoresis
Nylon mesh, 50, 12" x 12"	Ted Pellam, Inc	41-12105
GE Storage Phosphor Screens	GE Healthcare Life Sciences	28-9564-75
Phosphor Screen Exposure Cassette	GE Healthcare Life Sciences	63-0035-44
Isotemp Hybridzation Incubator	Fisher Scientific	13-247-20Q
Cylindrical hybridization tubes	Fisher Scientific	13-247-300
The Belly Dancer orbital shaker	Sigma-aldrich	Z768499-1EA	Orbital shaker
Typhoon 9400	ABI		For imaging of gels and phosphor screens
GraphPad Prism 6	GraphPad	Version 6.05	Graphing Program
Microsoft Excel	Microsoft	Office 365	Spreadsheet program
Nanodrop	Thermo Scientific	Nanodrp 2000	Spectrophotometer