How to cut DNA

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Sat Sep 21 08:42:43 EST 2002


On 21 Sep 2002 01:17:11 -0700, rajgupta121 at rediffmail.com (Raj) wrote:

>Hello ,
>
>  I am a physicist. I want to know how people cut DNA precisely to
>their required base pairs?

Usually we use a restriction enzyme (restriction endonuclease); there
are a variety of such enzymes that have been identified. The most
useful (the Type II, although the younger molecular biologists may not
realize that there are other types) are those that cut DNA at defined
sequences. For example, the enzyme Eco RI cuts DNA only at the
sequence GAATTC, between the G and A.

To search for enzymes or recognition sequences, you can use NEB's
REBASE:

<http://rebase.neb.com/rebase/>

If you want to cut at a specific site not defined by an available
restrtion enzyme, there are a few other ways. In the "olden days" we
might have had to use some kind of non-specific  exonuclease to trim
the DNA to approximately the right size, then brute-force screen the
products to find the right molecule. Nowadays, we[1] can make[2] short
pieces of DNA (oligonucleotides) of defined sequence; the "oligo" can
then be used as a primer to amplify the correct piece of DNA by the
Polymerase Chain Reaction (PCR).

> After cutting how they extract that base
>pair DNA?

Usually by gel electrophoresis, but other techniques can be used, such
as HPLC.

Nick

[1] By "we" I mean the DNA synthesis machine, or a biotech supply
company that has one.

[2] By "make" I mean filling out an order form and faxing it to the
place that has a DNA synthesizer.

-- 
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu



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