rapid and sensitive method for screening YAC libraries which involves screening of DNA from ... K acetate is added and the minipreps kept on ice for one hour.
Volume 17 17 Number Number 14 14 1989 1989
Volume
Nucleic Acids Research Nucleic Acids Research
An improved method for the screening of YAC libraries Edith Heard, Brendan Davies, Salvatore Feo and Mike Fried Eukaryotic Gene Organisation and Expression Laboratory, Imperial Cancer Research Fund, PO Box 123, Lincoln's Inn Fields, London WC2A 3PX, UK Submitted June 12, 1989
The cloning and mapping of large regions of genomic DNA has been greatly facilitated by Yeast Artificial Chromosome (YAC) vectors1. However, the yeast system causes certain problems In the screening of YAC libraries by colony hybridization: (1)Primary yeast transformants must be embedded in agar, which precludes direct colony screening. (2)The number of YACs per cell and the number of cells per yeast colony are much lower than in cloning in bacteria. (3)The spheroplasting of the colonies on the filters prior to lysis can be inefficient leading to under-representation of some colonies. Here we describe a rapid and sensitive method for screening YAC libraries which involves screening of DNA from pools of colonies using the Polymerase Chain Reaction (PCR)2. Primary yeast transformants are picked into 96 well microtitre dishes for library storage (0.2ml selective medium/well). DNA from the colonies grown up in each dish can be prepared using a rapid miniprep method, after pooling together 0.1 ml of saturated culture (5xlO5cells/well) from each of the 96 wells using a 12-channel micropipette. The pools are spun down, washed once in SCE buffer (1 M sorbitol, O.1M sodium citrate, 10mM EDTA), resuspended in 0.32 ml SCEM (SCE plus 30mM 2mercaptoethanol) and spheroplasted by incubation at 300C for about 30' with Lyticase (2.85units/ml). The spheroplasts are gently spun down (30" in microfuge), resuspended In 0.4 ml of TE containing 40mM EDTA, 75mM Tris (pH7.4),0.4% SDS, and lysed by incubation at 680C for 30'. 0.1 ml of cold 4M K acetate is added and the minipreps kept on ice for one hour. Precipitated protein is spun down (15' at 40C), the supernatant transferred to a fresh tube and the DNA is ethanol precipitated. The pellet is resuspended in 0.3 ml TE, treated with RNAase and then the DNA is reprecipitated, washed in 80% ethanol, and resuspended in IOX of TE (final conc. 0.2-0.5mg DNANml). Thus, DNA for screening can be prepared from 24 pools (representing 2304 colonies) simultaneously in just a few hours. The DNA from the pools is stored for subsequent screenings. For screening, 1 p1 of DNA from each of the pools is used in a single PCR using a pair of oligomers derived from a known target sequence as primers. If one of the 96 colonies represented in each PCR contains the sequence between the primers, a band of appropriate size will be amplified. The figure shows a number of pools screened for a YAC containing a region of Polyoma virus DNA intergrated into rat DNA, using 27 bp primers derived from Polyoma sequence that were about I kb apart. Only the pool containing this sequence amplifies the 1 kb fragment, giving a band on an ethidium bromide stained gel. In the event of inefficient PCRs (bad primers, poor growth of positive colony in well), a blot of this gel may allow detection of the band by hybridization. The positive colony is identified by PCRs on row and column minipreps of the appropriate microtitre dish, and this can be verified by PCR on the single (untreated) colony. 4 M 1 2 3 M M t 2 s t Fig.Legend: 1% agarose gel of PCR products from a selection of screened pools. Lane 2: Pool containing positive colony. Lanes land 3: Two random negative pools. Lane 4: PCR product from 2jg genomic rat DNA containing the target sequence. M: 123bp ladder markers.
Reference. 1. Burke et al (87), Science_= 806-812. 2. Saiki et a! (88), Science 2j 487-491. Q1% IRI I n L- PD %w rressC
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