Honghua Li

Analysis of DNA sequence variation in single cells

DNA sequences in single cells can be analyzed by the polymerase chain reaction. Detailed methods for the isolation, lysis, amplification, and detection of alleles in single cell samples are given, with an emphasis on protocols for individual sperm. The rationale used in primer design to maximize amplification efficiency and to enhance specificity and methods to control sample contamination are also discussed.

Ordering three DNA polymorphisms on human chromosome 3 by sperm typing.

Three loci on the short arm of human chromosome 3 were ordered by sperm typing to expand the limited genetic map of this region. Almost 300 individual sperm from a donor triply heterozygous at D3S2, D3S11, and D3S12 were amplified by PCR using primers flanking the polymorphic site at each locus. Primary PCR product was reamplified using allele-specific primers of different lengths, allowing the allelic state at each locus to be determined by gel electrophoresis. Maximum likelihood analysis of the sperm-typing data showed that the most likely order was D3S2-D3S11-D3S12 with an odds ratio of almost 5000:1 when compared to the next most likely order. This finding should be useful in interpreting loss of heterozygosity on 3p in a variety of cancers. Our results also demonstrate the practicality of ordering DNA polymorphisms using sperm typing.

Gene-centromere linkage mapping by PCR analysis of individual oocytes

We describe a general method of determining the recombination fraction between a polymorphic locus and the centromere in any species where single oocytes can be obtained. After removal of the first polar body, each oocyte is analyzed by PCR. The frequency of oocytes heterozygous at the polymorphic locus is used to estimate the recombination fraction. We estimate a recombination fraction of 0.15 between the mouse major histocompatibility complex (H-2) and the centromere of chromosome 17.

Sperm typing allows accurate measurement of the recombination fraction between D3S2 and D3S3 on the short arm of human chromosome 3

The interval between the D3S2 and D3S3 loci on human chromosome 3p is a frequent site of deletions in a number of different cancers and contains the most common fragile site in man. Both loci have been physically mapped to 3p but because heterozygosity for D3S3 is so infrequent, recombination between them could not be determined accurately by using family studies. Sperm typing can measure recombination between DNA polymorphisms even in a single individual and thus can make use of polymorphisms with a low PIC. The recombination fraction between D3S2 and D3S3 was estimated to be 0.28 based on analyzing 189 and 77 sperm from two doubly heterozygous donors, respectively. These results demonstrate one of the ways in which sperm typing can complement pedigree analysis in constructing genetic maps.

Single Sperm PCR Analysis — Implications for Preimplantation Genetic Disease Diagnosis

The polymerase chain reaction (PCR; Saiki et al., 1985, 1988; Mullis and Faloona, 1987) is a method of selective in vitro gene amplification. The principle of the PCR method is shown in Figure 1. Two small stretches of DNA of known sequence that flank the target region to be amplified (Figure la) are used to design two oligonucleotide primers. The synthetically made primers are chosen so that one is complementary to one flanking sequence while the other is complementary to the other flanking sequence. The 3’ hydroxyl ends of the primers face the target sequence. Following DNA denaturation of the target, the single stranded primers hybridize to their complementary flanking sequences (Figure lb). In the presence of a DNA polymerase the primers will be extended through the target sequence (Figure 1c). DNA denaturation, primer hybridization and DNA polymerase extension represent one PCR cycle. If the first cycle is followed by a second one (Figure 1d), more copies of the target sequence will be made.