PCR using Us instead of Ts
A.P.R.M. OSTEROP at TCH
OSTEROP at tch.fgg.eur.nl
Fri Jan 19 04:29:27 EST 1996
In article <199601182252.JAA05825 at morgan.angis.su.OZ.AU> nswema05 at angis.su.OZ.AU writes:
>From: nswema05 at angis.su.OZ.AU
>Subject: PCR using Us instead of Ts
>Date: 18 Jan 1996 14:54:17 -0800
>Us are often used in place of Ts for PCR reactions to allow Uracil-N-
>glycocylase to be used for removal of potential contamination with PCR
>products. However, PCR products containing Us are difficult to clone because
>upon transformation bacterial UNGs destroy the vector plus U-containing
>insert. In my opinion, the product information provided by companies
>marketing vectors for cloning PCR products should point out that PCR products
>containing Us are not suitable for cloning. This could create problems when
>a PCR product amplified using Us turns out to be of interest and it is
>desired to clone it. In this situation, I have tried to reamplify products
>using Ts, but on occasion have been thwarted when I had no more source DNA
>(from clinical specimens) and the U-containing PCR product was refractory
>to being reamplified using the same PCR primers (or even best-guess nested
>primers). To attempt to overcome this problem, I obtained an UNG negative
>strain of E coli, CJ236, and transformed competent bacteria with my
>U-containing PCR product inserted into a TA vector. Unfortunately, the
>plasmids recovered from this strain using several different methods of
>plasmid mini-prep appeared to be degraded.
>Does anyone have further suggestions for cloning U-containing PCR products?
>Do you feel that this potential problem has been inadequately publicised?
>I can see that considerable time could be wasted trying to clone PCR products
>that contain Us using standard methods.
Hi I am also into UDG. I have obtained a paper published by the Life Tech.
company (FOCUS 14(2) p. 53-56 latest reference is from 1992) in wich an ung
minus strain is tested. I do not have any experience with it myself. It is a
modified DH10B strain called 3138C wich has become 10 times less competent
in general using pUC19 and 20-40 times less using the ? pT7/T3 alfa 19 ?
vector. The 3138C strain is tested for ung minus properties by cloning an
UTP pcr product encoding the kanamycin resistance gene (1.3 kb) into pT7/T3
alfa 19 and subsequent transformation. The cells became Kan resistant. I
suppose the plasmid is not degraded in these cells. What happens upon mini-
prepping the plasmid I can not foresee but it should be no problem I guess.
The authors, R.L Bebee C.G. Thornton et al., thank J.J. Lin for
constructing and providing the competent 3138C strain but there is no
indication how to contact Lin.
Another topic in this paper is the efficiency of restriction enzymes to
recognize U containing sequences.
KpnI, NotI, SmaI do not contain U in the recognition site and do digest.
SalI, MluI, RsrII do digest.
EcoRI, HindIII, SphI, XbaI, SpeI, SstI do not digest however BamHI reacts
confused. One PCR product it does not cut while the other is cut. The
authors state that not only the recognition site is important but also the
aligning sequence especially if it is U rich. Thus you have to test the
enzyme of interest over again if you want to cut another PCR product.
Erasmus Univ. Rotterdam
osterop at tch.fgg.eur.nl
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