Telomere length determination protocol?

[Dan Bolser]

What is the cheapest way to measure my telomeres? (Average of white
blood cell telomere length).

Seems PCR is the way to go, but I'm starting from scratch. i.e.

* Where can I order the oligos (what sequence)?
** How should I store them?
* Where can I buy a PCR machine? (I don't want to build one).
* What is the best / cheapest way to measure the length of a PCR
product?
** I like the idea of capillary electrophoresis, but I guess that is
pricey ...


I spoke to Elizabeth Blackburn after she presented some of the work
described here:
http://groups.google.com/group/diybio/browse_thread/thread/5eedf6c1de29fc10

She mentioned several methods for measuring telomere length, but she
said the easiest is simple PCR.

I'd like to measure the length of my telomeres every few day for a
month or so.


Cheers,
Dan.

[Jonathan Street]

Would you expect the length of your telomeres to vary over a time period of just days?  I've not looked into it but my feeling would be any change would take longer.

It's been some time since I last ordered oligos so others will probably be better able to answer your first question. 
They are normally sent in a lyophilised (dried) form and can be left like that long term at room temp.  Once you dissolve them they should be stored at -20 degC.

Projects like openpcr (http://openpcr.org/) although offering kits I believe also plan to offer assembled thermal cyclers which will probably still work out to be cheaper than new commercial units.  http://otyp.es/ is also working on a thermal cycler which I believe will be assembled.

The standard way to measure PCR products is an agarose gel.  The main expenses are the tank and the power supply.  Capillary electrophoresis would certainly be very nice but I suspect you are right on the price.  It's required for very precise length determination so for example is often used for sequencing when you need to view each base separately.  I suspect telomeres lengthen in steps of greater than one base so agarose will probably work.

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[Cathal Garvey]

Telomeres are at least 6 bases per repeat, can't recall the human repeat offhand.

You may have to do something involving strong chelation to your extracted DNA to properly unfold telomeres, because if memory serves they fold into some complex, inaccessible form around metal ions

---
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On 12 Oct 2010 17:05, "Jonathan Street" <streetj...@gmail.com> wrote:

Would you expect the length of your telomeres to vary over a time period of just days?  I've not looked into it but my feeling would be any change would take longer.

It's been some time since I last ordered oligos so others will probably be better able to answer your first question. 
They are normally sent in a lyophilised (dried) form and can be left like that long term at room temp.  Once you dissolve them they should be stored at -20 degC.

Projects like openpcr (http://openpcr.org/) although offering kits I believe also plan to offer assembled thermal cyclers which will probably still work out to be cheaper than new commercial units.  http://otyp.es/ is also working on a thermal cycler which I believe will be assembled.

The standard way to measure PCR products is an agarose gel.  The main expenses are the tank and the power supply.  Capillary electrophoresis would certainly be very nice but I suspect you are right on the price.  It's required for very precise length determination so for example is often used for sequencing when you need to view each base separately.  I suspect telomeres lengthen in steps of greater than one base so agarose will probably work.

On 12 October 2010 15:12, Dan Bolser <dan.b...@gmail.com> wrote:
>

> What is the cheapest way to...

[Cory Tobin]

> * Where can I order the oligos (what sequence)?

IDT (www.idtdna.com) is the most popular one, although lots of
companies offer this service.

> ** How should I store them?

In TE (10mM Tris, 1mM EDTA, pH 8.0) at -20C

[ruphos]

* Where can I buy a PCR machine? (I don't want to build one).

University Surplus, eBay. We just got one on ebay for $300.

--
"And if ye cannot be saints of knowledge, then be at least its warriors."
-- Friedrich Nietzsche

[Cathal Garvey]

You can also store TE-dissolved DNA at 4C, or at -20 in PCR-grade, nuclease free water. Keep a working stock in the fridge and store the rest, don't freeze-thaw your DNA too often.

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To po...

[Dan Bolser]

On Oct 12, 5:05 pm, Jonathan Street <streetjonat...@gmail.com> wrote:
> Would you expect the length of your telomeres to vary over a time period of
> just days?  I've not looked into it but my feeling would be any change would
> take longer.

I don't know. I don't think it's been studied, which is why I'd like
to do it.

> It's been some time since I last ordered oligos so others will probably be
> better able to answer your first question.
> They are normally sent in a lyophilised (dried) form and can be left like
> that long term at room temp.  Once you dissolve them they should be stored
> at -20 degC.

Ahh... How about a freezer bag stuffed full of dry ice and kept in a
domestic freezer? Where can I get dry ice?

> Projects like openpcr (http://openpcr.org/) although offering kits I believe
> also plan to offer assembled thermal cyclers which will probably still work

> out to be cheaper than new commercial units.  http://otyp.es/is also

> working on a thermal cycler which I believe will be assembled.

Anything ready to buy now? What will costs be? Can't find any details
on those sites. I'll check the archives here.

> The standard way to measure PCR products is an agarose gel.  The main
> expenses are the tank and the power supply.  Capillary electrophoresis would
> certainly be very nice but I suspect you are right on the price.  It's
> required for very precise length determination so for example is often used
> for sequencing when you need to view each base separately.  I suspect
> telomeres lengthen in steps of greater than one base so agarose will
> probably work.

Yup, they are typically multiples of 6: TTAGGG

http://en.wikipedia.org/wiki/Telomere#Telomere_sequences


However, I'm not sure if this is the sequence one would use to PCR
them up. I was expecting to need a sub-telomeric sequence, but perhaps
I should research the protocol ;-)


Thanks for help,
Dan.

> On 12 October 2010 15:12, Dan Bolser <dan.bol...@gmail.com> wrote:
>
> > What is the cheapest way to measure my telomeres? (Average of white
> > blood cell telomere length).
>
> > Seems PCR is the way to go, but I'm starting from scratch. i.e.
>
> > * Where can I order the oligos (what sequence)?
> > ** How should I store them?
> > * Where can I buy a PCR machine? (I don't want to build one).
> > * What is the best / cheapest way to measure the length of a PCR
> > product?
> > ** I like the idea of capillary electrophoresis, but I guess that is
> > pricey ...
>
> > I spoke to Elizabeth Blackburn after she presented some of the work
> > described here:

> >http://groups.google.com/group/diybio/browse_thread/thread/5eedf6c1de...

>
> > She mentioned several methods for measuring telomere length, but she
> > said the easiest is simple PCR.
>
> > I'd like to measure the length of my telomeres every few day for a
> > month or so.
>
> > Cheers,
> > Dan.
>
> > --
> > You received this message because you are subscribed to the Google Groups
> > "DIYbio" group.
> > To post to this group, send email to diy...@googlegroups.com.
> > To unsubscribe from this group, send email to

> > diybio+un...@googlegroups.com<diybio%2Bunsu...@googlegroups.com>
> > .

[Dan Bolser]

On Oct 12, 5:26 pm, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Telomeres are at least 6 bases per repeat, can't recall the human repeat
> offhand.

From WP (http://en.wikipedia.org/wiki/Telomere):

"In humans, [the] telomere sequence is a repeating string of
TTAGGG, between 3 and 20 kilobases in length. There are an additional
100-300 kilobases of telomere-associated repeats between the telomere
and the rest of the chromosome."


I'm not sure if I need to prime in this telomere-associated repeat
region.

> You may have to do something involving strong chelation to your extracted
> DNA to properly unfold telomeres, because if memory serves they fold into
> some complex, inaccessible form around metal ions

From WP (http://en.wikipedia.org/wiki/Telomere):

"These G-rich sequences can form four-stranded structures (G-
quadruplexes), with sets of four bases held in plane and then stacked
on top of each other with either a sodium or a potassium ion between
the planar quadruplexes."


However, I don't think this is the 'natural' state for telomeres in
cells. Mostly they are protected by protein, which should be removed
during the DNA purification stage.

> ---
> Twitter: @onetruecathal
> Sent from my beloved Android phone.
>

> On 12 Oct 2010 17:05, "Jonathan Street" <streetjonat...@gmail.com> wrote:
>
> Would you expect the length of your telomeres to vary over a time period of
> just days?  I've not looked into it but my feeling would be any change would
> take longer.
>
> It's been some time since I last ordered oligos so others will probably be
> better able to answer your first question.
> They are normally sent in a lyophilised (dried) form and can be left like
> that long term at room temp.  Once you dissolve them they should be stored
> at -20 degC.
>
> Projects like openpcr (http://openpcr.org/) although offering kits I believe
> also plan to offer assembled thermal cyclers which will probably still work

> out to be cheaper than new commercial units.  http://otyp.es/is also

> working on a thermal cycler which I believe will be assembled.
>
> The standard way to measure PCR products is an agarose gel.  The main
> expenses are the tank and the power supply.  Capillary electrophoresis would
> certainly be very nice but I suspect you are right on the price.  It's
> required for very precise length determination so for example is often used
> for sequencing when you need to view each base separately.  I suspect
> telomeres lengthen in steps of greater than one base so agarose will
> probably work.
>

[Dan Bolser]

What do people do for -20?

[Jonathan Street]

For -20 a domestic freezer should be fine.  The only difference I'm aware of to a 'lab-grade' freezer is that the freezers in labs are certified as spark free.

If you wanted to go colder using dry ice I would use a small polystyrene box or a thermos with a small hole drilled in the lid to avoid pressure build up.

As far as I know the thermal cycler projects I mentioned aren't ready for retail yet.  No reason why you couldn't be collecting samples while you wait though.  I think the prices for an assembled instrument were going to be a little above $1000.  You might be able to get a second hand one on ebay for a similar price but it can be difficult to judge exactly what you're getting.  I think someone on the list put together a guide at some point.

For the protocol wikipedia is unlikely to go into the detail you need.  Openwetware.org might have a protocol but your best option will be a scientific article.  An article should contain all the info you need to repeat their experiment (in theory) including primer sequence.  If you're not affiliated with an institution and are struggling to access an article a quick message to the list should get a result fairly quickly.

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[Cathal Garvey]

Something to consider: priming within such a repetitive region with primers based on the repetitive element will give nonsense results, most likely. You'll get priming within the region, priming between products and probably some acrobatic self-priming. You'll probably get the latter two anyway to some extent, but to minimise the former you should aim to prime from outside.

You could possibly invent some 'fun' protocol involving covelently joining a reverse-priming tag to the end of your sequence with some kinase and a 3' recessed, telomere-compatible restriction site overhang. That way (if it worked) you could prime from both ends of the telomere without using the telomeric repeat element in your primers.

On 14 Oct 2010 10:03, "Jonathan Street" <streetj...@gmail.com> wrote:

For -20 a domestic freezer should be fine.  The only difference I'm aware of to a 'lab-grade' freezer is that the freezers in labs are certified as spark free.

If you wanted to go colder using dry ice I would use a small polystyrene box or a thermos with a small hole drilled in the lid to avoid pressure build up.

As far as I know the thermal cycler projects I mentioned aren't ready for retail yet.  No reason why you couldn't be collecting samples while you wait though.  I think the prices for an assembled instrument were going to be a little above $1000.  You might be able to get a second hand one on ebay for a similar price but it can be difficult to judge exactly what you're getting.  I think someone on the list put together a guide at some point.

For the protocol wikipedia is unlikely to go into the detail you need.  Openwetware.org might have a protocol but your best option will be a scientific article.  An article should contain all the info you need to repeat their experiment (in theory) including primer sequence.  If you're not affiliated with an institution and are struggling to access an article a quick message to the list should get a result fairly quickly.

On 14 October 2010 09:05, Dan Bolser <dan.b...@gmail.com> wrote:
>
>
>

> On Oct 12, 6:14 pm, Cor...

[Jonathan Street]

This paper (open access) presents one protocol for measuring telomere length: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC115301/?tool=pubmed

I found that referenced in this paper: http://www.ncbi.nlm.nih.gov/pubmed/20839490
That suggests you may well see changes in the telomere length over short periods of time as different cell populations are recruited to the circulation.

--

[jlund256]

>> They are normally sent in a lyophilised (dried) form and can be left like
>> that long term at room temp. Once you dissolve them they should be stored
>> at -20 degC.
>

> Ahh... How about a freezer bag stuffed full of dry ice and kept in a
> domestic freezer? Where can I get dry ice?

Dry ice isn't needed and isn't an easy long-term solution. The
primers will keep in an ordinary freezer. The colder the longer
they'll keep; they'll be fine for 1+ year in an ordinary freezer. The
important thing is to put the primers (and associated reagents) inside
a airtight container inside the freezer. In a typical defrosting
freezer ice slowly evaporates and you need to prevent this.

Jim Lund

[helicase]

Use a freezer that's not frost-free. The freeze/thaw cycles in frost-
free freezers are undesirable for your samples and reagents!

[Phil]

On Oct 14, 4:05 am, Dan Bolser <dan.bol...@gmail.com> wrote:
> On Oct 12, 5:26 pm, Cathal Garvey <cathalgar...@gmail.com> wrote:
>
> > Telomeres are at least 6 bases per repeat, can't recall the human repeat
> > offhand.
>
> From WP (http://en.wikipedia.org/wiki/Telomere):
>
>     "In humans, [the] telomere sequence is a repeating string of
> TTAGGG, between 3 and 20 kilobases  in length. There are an additional
> 100-300 kilobases of telomere-associated repeats between the telomere
> and the rest of the chromosome."

3 kilobases is too long to sequence in a single read with any
sequencing technology I know. And if it's a repeat, you can't shear
the DNA and sequence the fragments. How can you sequence a repeat 3kb
long, with any technology at all?

You want to amplify with some primer that is NOT part of the repeat.
But if the repeat is the very end of the chromosome, then there is no
possible primer on the other side of the repeat.

How do people approach this problem?

The answer might not involve PCR at all. See if you can find a
collection of restriction enzymes such that the combination of them
leave no sequences over 1kb uncut in the human genome, except for the
telomeres. Then isolate a bunch of your DNA, chop it up wi the
enzymes, and run it on a gel.