Andrew Thliveris

Identification and characterization of the familial adenomatous polyposis coli gene

DNA from 61 unrelated patients with adenomatous polyposis coli (APC) was examined for mutations in three genes (DP1, SRP19, and DP2.5) located within a 100 kb region deleted in two of the patients. The intron-exon boundary sequences were defined for each of these genes, and single-strand conformation polymorphism analysis of exons from DP2.5 identified four mutations specific to APC patients. Each of two aberrant alleles contained a base substitution changing an amino acid to a stop codon in the predicted peptide; the other mutations were small deletions leading to frameshifts. Analysis of DNA from parents of one of these patients showed that his 2 bp deletion is a new mutation; furthermore, the mutation was transmitted to two of his children. These data have established that DP2.5 is the APC gene.

Identification of deletion mutations and three new genes at the familial polyposis locus

Small (100-260 kb), nested deletions were characterized in DNA from two unrelated patients with familial adenomatous polyposis coli (APC). Three candidate genes located within the deleted region were ascertained and a previous candidate gene, MCC, was shown to be located outside the deleted region. One of the new genes contained sequence identical to SRP19, the gene coding for the 19 kd component of the ribosomal signal recognition particle. The second, provisionally designated DP1 (deleted in polyposis 1), was found to be transcribed in the same orientation as MCC. Two other cDNAs, DP2 and DP3, were found to overlap, forming a single gene, DP2.5, that is transcribed in the same orientation as SRP19.

Identification of Intron/Exon Boundaries in Genomic DNA by Inverse PCR

This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template. This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA.

Deriving Probes From Large‐Insert Clones by PCR Methods

This unit describes several polymerase chain reaction (PCR)‐based methods to obtain DNA fragments from clones with large inserts without prior knowledge of the insert DNA sequence. The protocols can be categorized into three groups: (1) methods to generate DNA fragments at random representing the entire length of the cloned insert, (2) methods to generate DNA fragments representing the extremities of an insert, and (3) methods to generate complex probes suitable for fluorescence in situ hybridization. Support protocols describe direct cloning of these PCR products and the isolation of total yeast DNA from yeast artificial chromosome (YAC) clones.