Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.

Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species.

The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment. Tidiness and adherence to a strict set of protocols can avoid disaster.

Avoiding false positives with PCR

Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.

Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics

The identification of predefined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. Here, we describe an approach for transforming the exponential, analog nature of the PCR into a linear, digital signal suitable for this purpose. Single molecules are isolated by dilution and individually amplified by PCR; each product is then analyzed separately for the presence of mutations by using fluorescent probes. The feasibility of the approach is demonstrated through the detection of a mutant ras oncogene in the stool of patients with colorectal cancer. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.

https://www.pnas.org/content/pnas/96/16/9236.full.pdf

Digital PCR

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Because DNA yield from human samples is frequently limiting, much effort has been invested in developing methods for whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. However, existing WGA methods like degenerate oligonucleotide-primed PCR suffer from incomplete coverage and inadequate average DNA size. We describe a method, termed multiple displacement amplification (MDA), which provides a highly uniform representation across the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from biological samples including crude whole blood and tissue culture cells. MDA-amplified human DNA is useful for several common methods of genetic analysis, including genotyping of single nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and reliable method that could have significant implications for genetic studies, forensics, diagnostics, and long-term sample storage.

https://www.pnas.org/content/pnas/99/8/5261.full.pdf

Comprehensive human genome amplification using multiple displacement amplification

We have developed an in vitro method for amplifying a large fraction of the DNA sequences present in a single haploid cell by repeated primer extensions using a mixture of 15-base random oligonucleotides. We studied 12 genetic loci and estimate that the probability of amplifying any sequence in the genome to a minimum of 30 copies is not less than 0.78 (95% confidence). Whole genome amplification beginning with a single cell, or other samples with very small amounts of DNA, has significant implications for multipoint mapping by sperm or oocyte typing and possibly for genetic disease diagnosis, forensics, and the analysis of ancient DNA samples.

https://www.pnas.org/content/pnas/89/13/5847.full.pdf

Whole genome amplification from a single cell: implications for genetic analysis

The use of the polymerase chain reaction for analysing DNA sequences in individual diploid cells and human sperm shows that two genetic loci can be co-amplified from a single sperm, which may allow the analysis of previously inaccessible genetic phenomena.

Amplification and analysis of DNA sequences in single human sperm and diploid cells

The frequency of recombination between the G gamma-globin (HBG2) and parathyroid hormone (PTH) loci on the short arm of human chromosome 11 was estimated by typing greater than 700 single-sperm samples from two males. The sperm-typing technique employed involves the polymerase chain reaction and allele-specific oligonucleotide hybridization. Our maximum likelihood recombination fraction estimate of 0.16 (95%) confidence interval, 0.13-0.19) falls well within previous estimates based on family studies. With current technology and a sample size of 1000 sperm, recombination fractions down to approximately 0.009 can be estimated with statistical reliability; with a sample size of 5000 sperm, this value drops to about 0.004. Reasonable technological improvements could result in the detection of recombination frequencies less than 0.001.

https://www.pnas.org/content/pnas/86/23/9389.full.pdf

Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers

We have developed a procedure that allows the detection of polymerase chain reaction (PCR) products derived from a single target DNA molecule in a human sperm without using radioactive probes. With this method, three genetic loci present in a single sperm can be amplified simultaneously. The amplification procedure is specific as well as efficient and permits detection of the PCR product by ethidium bromide staining after polyacrylamide gel electrophoresis. When allele-specific PCR primers that differ in length are used, the size of the PCR products of different alleles also vary in length, allowing the allelic state at each locus to be determined electrophoretically. Studies on individual sperm by using this procedure should facilitate the measurement of genetic recombination in humans over small physical distances. The ability to directly analyze the allelic state of PCR products from one cell rapidly and simply will also be useful for the prenatal diagnosis of genetic disease, especially in the analysis of single blastomeres taken from in vitro fertilized eggs prior to implantation.

https://www.pnas.org/content/pnas/87/12/4580.full.pdf

Xiangfeng Cui

Papers

Whole genome amplification from a single cell: implications for genetic analysis

Amplification and analysis of DNA sequences in single human sperm and diploid cells

Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers

Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction.

Analysis of DNA sequence variation in single cells