Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells.
The polymerase chain reaction technique is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. However, some investigators need to sequence the entire coding region of mammalian genes, e.g., cellular ras genes or the gene encoding hypoxanthine (guanine) phosphoribosyl transferase (HPRT), to determine what specific changes have occurred. To do so, they isolate RNA from large populations of cells, amplify cDNA from the gene of interest, subclone the product, and sequence two or more isolates to determine the common mutation. We have developed a method to simplify this procedure by copying mRNA of the hprt gene directly from the lysate of a clone of mutant diploid human fibroblasts (e.g., 100 cells). We amplified the first and second strand of the cDNA of the gene of interest 10(10)- to 10(11)-fold, obtained 5 to 10 micrograms of DNA in less than 10 h, and sequenced the coding region directly without the need for RNA extraction or DNA template purification. By our method cDNA can be amplified directly from the lysate of just one human cell, but to avoid detecting random changes introduced by the polymerase, we lysed approx. 200 cells from a clone, each containing the identical mutation, amplified the cDNA, and determined the consensus sequence by direct nucleotide sequencing.
Yang
J L
JL
Department of Microbiology, Michigan State University, East Lansing, MI 48824.
Maher
V M
VM
McCormick
J J
JJ
D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004171:Diploidy; D011905:Genes, ras; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012867:Skin / Q000378:metabolism; D013698:Templates, Genetic
D012333:RNA, Messenger; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase