Multiplex PCR from hairs

David MacHugh dmachugh at mail.tcd.ie
Mon Mar 9 07:52:35 EST 1998



Hi, someone may be able to shed some light on a problem we have been
having with multiplex PCR of microsatellites using DNA from cattle hairs.

We are using genomic DNA prepared from hair samples (approx. 5 hair
roots).  Each hair root is clipped as close to the root as possible to
minimise the amount of actual hair in the sample.  The roots are treated
with 50µl of 0.2 M NaOH for 20 mins at 96C.  Then 50µl of the following
solution is added:

200mM HCl
100mM Tris-HCl pH8.5

These DNA samples work fine for normal PCR with a single set of primers. 
However, when a well-established multiplex system is used, the results are
very disapointing.  The multiplex works well with normal genomic DNA
isolated from blood using standard ProtK/organic extraction methods.

Does anyone have any suggestions as to how the multiplex results could be
optimized using the hair root DNA samples? - an alternative quick
extraction method? extra purification steps? changes to the enzyme
concentration? modifications to the PCR buffer (extra KCL)? etc...

We are using the following equipment:

MJ Research PTC200 thermocycler for the PCR amplifications and a LICOR
4200 automated genotyper for the analysis.


Thanks in advance.


David MacHugh Ph.D.
Genetics Department,
Trinity College,
Dublin 2.
PH: 353-1-608-2719
FAX: 353-1-679-8558
email: dmachugh at tcd.ie



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