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[bio.dros] single embryo PCR

shuji hanai shanai at md.tsukuba.ac.jp
Wed Jul 22 16:07:55 EST 1998


Dear Dr. Cohen and Fly Workers,

I made a protocol for PCR to detect deletion of interested gene.
I found a note by Dr. Garozzo et al.(DIN 13) for DNA preparation from single embryo. And Dr. Grevelding et al. reported direct PCR from whole embryo without DNA prep(1996 Nuc. Ac. Res. 24 4100-1). However, I could not find PCR applied to detect deletion.

I'm preparing a manuscript of my protocol applied to a gene. Could you tell me if you know any report using same method, especially PCR after staining for beta-galactosidase. I can send detailed information about my protocol, if needed.

My strategy is adaptable not only to detect deletion but also selected amplification of mutated allele such as EMS-mutants witch are lethal at embryo. I balanced deletion mutants with a blue balancer. Embryo with homozygous deletion can be identified by staining for beta-gal. Lysate of single embryos was prepared after the staining. Primers for an internal control(ftz in my case) and for interested gene(Poly(ADP-ribose) polymerase) were used for PCR. Beta-gal negative embryos gave no amplification of PARP gene but ftz. Needless to say, formaldehyde is toxic to DNA, but 500 bp can be amplified in my case.

Identification using blue balancer is a standard method, but PCR after fixation with formaldehyde and staining for beta-gal is not reported.

Without blue balancer, it's possible to estimate deletion by PCR from many single embryos, but it's not conclusive. However, direct PCR from whole embryo may be possible, you have to prepare embryo lysate for control and interested gene, except multiprimed PCR. Using my protocol, result may be decisive and DNA preparation from single embryo is not essential.

At 16:09 98.7.21 +0000, Merav Cohen wrote:
>Does anybody happen to fortuitously have a reference to a protocol or 
>an article describing single embryo PCR used to map a gene to a 
>deficiency/deletion?
Shuji Hanai
Dept. of Biochem. Inst. of Basic Med. Sci. Univ. of Tsukuba
1-1-1, Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Phone; +81 298 53 3272, FAX; +81 298 53 3271
shanai at md.tsukuba.ac.jp




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