Re: [DIYbio] Can anyone tell me what kind of equipment and supplies I would need to look at telomeres in cells?

On Tue, Jun 23, 2015 at 5:50 PM, Brad S <bradesnyder12@gmail.com> wrote:
> Can anyone tell me what kind of equipment and supplies I would need to look
> at telomeres in cells?

If you actually want to 'look' and see an image, you'll need to do
some heavy-metal staining of the DNA (look up osmium stain, uranyl
stain), then comb it out, then image under electron microscopy. A
decent SEM (scanning electron microscope) would probably be sufficient
to see the length of the DNA strands, but you won't know when the
sequence changes (when does the telomere section start). I don't know
if there's some enzyme that sticks to that interface, to give you a
marker for the start (to then find the total length).

Read up on the Halcyon Molecular team's publications (they were trying
to do sequencing with TEM). That company shutdown without making
commercial success, I think the main problem was the electron beam for
imaging was tearing apart the DNA strand.


If you don't want to actually 'look' at the telomeres, but rather
analyze them... then it seems the TRAP assay is indeed an option (I
don't know what all the options are). From this paper:
http://www.sciencedirect.com/science/article/pii/S0014579300023772

it says:

2.9. Detection of telomerase activity
Telomerase activity was measured using a PCR-based telomeric repeat
ampli¢cation protocol (TRAP) enzyme-linked immunosorbent assay (ELISA)
kit (Boehringer Mannheim) according to the manufacturer's description
with some modi¢cations. In brief, approximately 1U106 cells were lysed
in 200 Wl lysis reagent and incubated on ice for 30 min. For
conducting TRAP reaction, 2 Wl of cell extract (containing 2 Wg
protein) was added to 25 Wl of reaction mixture and then an Fig. 4.
E¡ect of GSH on OH-induced apoptosis in HeLa cells as detected by the
TUNEL procedure, £ow cytometric analysis, and DNA laddering assay.
Cells were preincubated with GSH for 1 h before a 24 h OH treatment.
A: Percentage of TUNEL positive cells. B: Percentage of Sub-G1 cells.
Values are the means þ S.D. of three independent experiments done in
triplicates. Error bars indicate standard deviations. C: DNA laddering
in cells pretreated with GSH followed by 0.1 mM FeSO4/0.6 mM H2O2
treatment. Lane 1: markers; lane 2: control; lane 3: DNA from
OH-treated cells; lanes 4^6: DNA from cells pretreated with 1 mM, 3
mM and 5 mM GSH, respectively. FEBS 24468 12-1-01 126 J.-G. Ren et
al./FEBS Letters 488 (2001) 123^132 appropriate amount of sterile
water was added to make a ¢nal volume of 50 Wl. PCR was performed in a
PTC-1001 Programmable Thermal Controller (MJ Research, Inc.) as
follows: primer elongation (30 min, 25³C), telomerase inactivation (5
min, 94³C), product ampli¢cation by repeat of 30 cycles (94³C for 30
s, 50³C for 30 s, 72³C for 90 s). Hybridization and ELISA reaction
were carried out following manufacturer's instruction. The extract
from normal human ¢broblasts served as negative controls. The extract
of 293 cells was used as positive controls.

2.10. Measurement of telomere length
Fluorescence in situ hybridization was carried out following the
procedure described by Hultdin et al. [20] and Rufer et al. [21] with
some modi¢cations. Trypsinized HeLa cells were harvested and washed
with ice-cold PBS, centrifuged at 500Ug for 5 min and resuspended in 1
ml PBS. Typically, 5U105 cells (each sample was divided equally into
two Eppendorf tubes) were used per tube. After centrifugation at
4900Ug for 30 s, the pellets were resuspended in a hybridization
mixture containing 70% formamide (Fluka BioChemika, Buchs,
Switzerland), 1% blocking reagent(Boehringer Mannheim GmbH, Mannheim,
Germany) and £uorescein-(CCCTAA)3-£uorescein PNA probe synthesized
using the Expedite 8909 Nucleic Acid Synthesis System (PerSeptive
Biosystems, Framingham, MA, USA) in 10 mM Tris pH 7.2. The volume of
the hybridization mixture was adjusted to 100 Wl/105 cells. Samples
were heated for DNA denaturation for 10 min at 82³C followed by
hybridization in the dark at room temperature overnight, the cells
were centrifuged and washed twice in PBS at 40³C. Cells were then
resuspended in PBS containing RNase A at 10 Wg/ml (Boehringer
Mannheim) and propidium iodide at 0.1 Wg/ml, vortexing and incubated
for 2^4 h at room temperature and analyzed immediately with a Coulter
Elite Flow Cytometer or stored at 4³C for up to 2 days prior to
analysis. For £ow cytometric analysis, the FL1 channel was used for
detection of £uorescein signal and the FL3 channel was for propidium
iodide. List mode data from 1U104 cells in each experiment were
collected and analyzed using Coulter Elite workstation 4.0 software
(Coulter Corp.). The telomere £uorescence signal was de¢ned as the
mean £uorescence signal in cells after substraction of the background
£uorescence signal.

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