Sequencing single-copy sequences

The DNA in a eukaryotic genome can be divided into different sequence classes. These are unique-sequence, or nonrepetitive, DNA and repetitive-sequence DNA. In the haploid genome, unique-sequence DNA generally includes the single copy genes that code for proteins. The repetitive DNA in the haploid genome includes sequences that vary in copy number from two to as many as 10 copies per cell. 

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ahility to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1-10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13). 

Ray, R., Komminoth, P., Machado, M., and Wolfe, H. J. (1991) Combined polymerase chain reaction and in situ hybridization for the detection of single copy genes and viral genomic sequences in intact cells. Mod. Pathol. 4,124A. 

Figure 2.6. The use of the split synthesis technique to generate a dipeptide library. After the two coupling steps shown are completed, the four possible (2 ) dipeptide sequence combinations are found. Note that any single bead will have multiple copies of the (identical) dipeptide attached and not just a single copy as shown above. This synthesis technique is economical and straightforward to undertake and requires no sophisticated equipment. The coupling steps, etc. utilized are based upon standard peptide S5mthesizing techniques, such as the Merrilield method (Box 2.1)...

The receptors bind to the cognate HRE mainly as dimers, allowing the formation of homodimers as well as heterodimers between various receptor monomers. We know of very few nuclear receptors whose HRE contains only a single copy of the recognition sequence. These receptors bind as monomers to the cognate HRE. 

The nicotinic acetylcholine receptor has five subunits single copies of subunits /3, y, and 8, and two identical a subunits each with an acetylcholine-binding site. All five subunits are related in sequence and tertiary structure, each having four transmembrane helical... 

Chloroplasts contain large 120- to 169-kb circular genomes encoding about 100 proteins (Chapter 23). A characteristic feature of most chloroplast DNA is the presence of long inverted repeat sequences (10,058 bp in the liverwort, 25,339 bp in tobacco).463 464 These are separated by 19,813 and 81,095 bp single copy regions in the liverwort and by similar sized regions in tobacco. Plastid DNA exists as a mixture of monomeric molecules with smaller amounts of dimers, trimers, and tetramers.464... 

For repetitive DNA sequences, such as alphoid repeats, especially for identification of centromeric regions on chromosomes, as well as in mterphase nuclei, I routinely prepare labeled probes by PCR using a centromeric DNA-specific set of primers. For single-copy gene mapping by FISH, it is essential to use DNA cloned into either cosmids or in yeast artificial chromosome vectors (YAC) m order to obtain the level of sensitivity that will enable visualization of signal with a fluorescence microscope... 

In prokaryotes there is only one copy of DNA per cell, and in nearly all cases there is only a single copy of a gene. Apart from regulatory DNA and signalling sequences, there are very few silent, or non-translated DNA sequences, in prokaryotes. Moreover, each gene is typically collinear with the amino-acid sequence for which it codes. This is in contrast to the organisation of eukaryotic DNA which is structurally and functionally far more complex the structural parts of genes are split by silent DNA and there are multiple copies of the chromosomes. 

Single-copy DNA. A region of the genome the sequence of which is present only once per haploid complement. 

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