(Normalized cDNA libraries)

Technical Report · Tue Jan 01 04:00:00 EST 1991

OSTI ID:6153132

Weissman, S M

Four project are currently underway. First, we are generating a set of Not1 linking clones for analysis of the X chromosome. 105 different linking clones have been isolated, which is believed to be 60--90% of the total number of linking clones in the library. The average size of each clone is slightly more than one megabase. Second, we have applied the Polymerase Chain Reaction technique to generate deeper libraries of linking clones and also jumping clones. This technique offers important advantages over conventional jumping, such as the reduced DNA requirement. This technique can be used to generate chromosome specific linking (and possibly jumping) clones from sorted chromosomes. Third, to enrich for specific DNA fragments we initially used DNA probes and recA mediated homology searches, but in the past few years we've looked for an alternative selection approach. The technique developed has been to construct a sandwiched probe in which a short stretch of single stranded RNA links the biotinylated nucleic acid to the large fragment we wish to recover. Limited ribonuclease treatment gives a highly selective and very mild release step. Our major project has been to develop DNA libraries where all cDNAs are present in equal copy numbers. Since two-thirds of the mRNA in a representative cell is many copies of a small number of genes, normalizing all cDNAs can give a greater than 30-fold enrichment of low copy number genes. If the source of cDNAs is not a single cell, but rather a tissue of organism, normalization gives considerably greater enrichment. One approach to normalization describes is self-annealing, followed by a hydroxyapatite column to separate single stranded and doublestranded nucleic acids. Normalized libraries for human spleen and thymes are being developed. (MHB)

• Research Organization: Yale Univ., New Haven, CT (USA). School of Medicine
• Sponsoring Organization: DOE/ER
• DOE Contract Number: FG02-88ER60687
• OSTI ID: 6153132
• Report Number(s): DOE/ER/60687-T1; ON: DE91008513
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
550400 -- Genetics
59 BASIC BIOLOGICAL SCIENCES
BODY
CELL CONSTITUENTS
CHROMOSOME SORTING
CHROMOSOMES
CLONING
COSMIDS
DNA HYBRIDIZATION
DNA-CLONING
DOCUMENT TYPES
ENZYMES
ESTERASES
GENES
GENETIC MAPPING
HETEROCHROMOSOMES
HYBRIDIZATION
HYDROLASES
LYMPHATIC SYSTEM
MAPPING
MESSENGER-RNA
MOLECULAR BIOLOGY
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PHOSPHODIESTERASES
PLASMIDS
PROGRESS REPORT
RNA
RNA-ASE
SPLEEN
THYMUS
X CHROMOSOME