Real-time PCR assay for quantitative mismatch detection.

We describe here a quantitative real-time PCR assay for the detection of single-base-pair differences that does not require fluorescently labeled gene-specific probes or complicated primer combinations. Following PCR or RT-PCR of a gene segment that may contain allele-specific differences, 100 pg amplified product are used for a real-time PCR with allele-specific primers and SYBR Green. The use of HEPES buffer at a pH of 6.95 together with AmpliTaq DNA polymerase results in a threshold difference between the correct template and the mismatched template of as many as 20 cycles, depending on the mismatch. Correct matches can be detected in an excess of mismatched template at least at the 0.01 level for the six primer-template matches versus mismatches tested: GC vs. A.C, AT vs. G.T, GC vs. C.C, GC vs. G.G, AT vs. C.T, and GC vs. G.A. Because the initial amplification is separate from real-time detection, conditions can be independently optimized for each step, making the assay particularly suitable for the detection of allele-specific expression in single cells.

Quantitative RT-PCR-based analysis of allele-specific gene expression.

F1 hybrids resulting from intercrosses of inbred strains have provided an invaluable tool for the study of imprinting. The hybrids can be used to analyze parent-of-origin differences in expression of any gene, provided sequence differences exist between the two parental alleles. Methods used to detect allele-specific expression include ribonuclease protection assays (1) and allele-specific RNA in situ hybridization (2), as well as a number of reverse transcriptase polymerase chain reaction (RT-PCR)-based assays (see, for example, refs. 3 and 4). We describe here two such assays that are quantitative and require only single base differences between the two alleles. Both assays rely on the amplification of the RNA of interest by RT-PCR using primer sets that flank the sequence polymorphism, a method shown previously to yield amplicons whose allelic ratio is proportional to the ratio in the starting material, regardless of the number of cycles of amplification (5).

Use of terminal transferase-dependent antisense RNA amplification to determine the transcription start site of the Snrpn gene in individual neurons.

We describe here a very sensitive technique for RNA structure analysis and the determination of transcription start sites and demonstrate its use for mapping the start site of the imprinted Snrpn gene in individual hippocampal neurons. The method is adapted from reverse transcription-terminal transferase-dependent PCR (RT-TDPCR) to include amplification of the antisense sequence by in vitro transcription just prior to the final PCR step. The method should be useful for analysis of all genes for which variation in promoter usage and/or differences in RNA secondary structure may be specific to a given cell type or developmental stage.

Methylated DNA sequences in genomic imprinting.

Genomic imprinting is a special form of epigenetic system that determines the parent-of-origin-specific, or monoallelic, expression of a small number of genes, termed "imprinted" genes. Considerable sequence and methylation analysis of imprinted genes has revealed a common theme: Regions of allele-specific methylation inherited from the gametes, or primary differentially methylated regions (DMRs), are associated with CpG islands and repeat elements, and this overall structure suggests functional significance. For at least three imprinted genes the sequence of the primary DMR constitutes an element able to regulate gene activity in cis--a chromatin insulator and a promoter of an antisense transcript. In these cases the unique feature of imprinting appears to be in the ability to switch the regulatory capacity of these elements on or off by the absence or presence of inherited methylation. Increasing evidence therefore suggests that genomic imprinting for at least some genes constitutes the regulation of gene regulatory elements by methylation. An important challenge now is to determine how the differential methylation of primary DMR sequences is established in the germ line. If methylation is the primary imprint, then the processes establishing it are the primary imprinting mechanisms. Trans-acting factors that are expressed in one sex of germ line and not the other are likely to be involved, and their ability to methylate may be mediated through repeat elements associated with the sequence of primary DMRs.

Use of a reverse transcriptase-polymerase chain reaction assay to analyze allele-specific expression in individual hippocampal neurons.

We report here a single-cell RT-PCR assay for allele-specific gene expression that can be used to probe for somatic variability within the CNS. Such variability, arising from epigenetic (nonmutational) events or somatic mutation early in development, may give clues as to clonal origin and may also affect the inheritance pattern of some CNS disorders. As a model system, we used reciprocal F1 hybrids of the cross Mus musculus C57BL/6J x Mus musculus castaneus. RNA was isolated from individual dissociated pyramidal neurons from hippocampi of F1 pups. For each gene of interest, single base polymorphisms were identified between the two parental strains by automated sequencing of RT-PCR products. Allele-specific expression was then analyzed by means of the previously described quantitative RT-PCR single nucleotide primer extension (SNuPE) assay (Singer-Sam et al., PCR Methods Appl. 1:160-163, 1992). Individual neurons showed monoallelic expression of the two control genes, X-linked Rps4, and the imprinted gene Snrpn; in contrast expression of Ncam and F3cam, coding for neural cell adhesion molecules, was found to be biallelic.

Differential replication timing of X-linked genes measured by a novel method using single-nucleotide primer extension.

The ratio of two differentially replicating alleles is not constant during S phase. Using this fact, we have developed a method for determining allele-specific replication timing for alleles differing by at least a single base pair. Unsynchronized cells in tissue culture are first sorted into fractions based on DNA content as a measure of position in S phase. DNA is purified from each fraction and used for PCR with primers that bracket the allelic difference, amplifying both alleles. The ratio of alleles in the amplified product is then determined by a single nucleotide primer extension (SNuPE) assay, modified as described [Singer-Sam,J. and Riggs,A.D. (1993) Methods Enzymol., 225, 344-351]. We report here use of this SNuPE-based method to analyze replication timing of two X-linked genes, Pgk-1 and Xist, as well as the autosomal gene Gabra-6. We have found that the two alleles of the Gabra-6 gene replicate synchronously, as expected; similarly, the active allele of the Pgk-1 gene on the active X chromosome (Xa) replicates early relative to the silent allele on the inactive X chromosome (Xi). In contrast, the expressed allele of the Xist gene, which is on the Xi, replicates late relative to the silent allele on the Xa.

In vivo ultraviolet and dimethyl sulfate footprinting of the 5' region of the expressed and silent Xist alleles.

The Xist (X inactive specific transcript) gene plays an essential role in X chromosome inactivation. To elucidate the mechanisms controlling Xist expression and X inactivation, we examined in vivo DNA-protein interactions in the Xist promoter region in a female mouse cell line (BMSL2), which has distinguishable Xist alleles. In vivo footprinting was accomplished by treatment of cells with dimethyl sulfate or ultraviolet light, followed by ligation-mediated polymerase chain reaction of purified DNA. The expressed allele on the inactive X chromosome and the silent allele on the active X chromosome were separated by the use of a restriction fragment length polymorphism prior to ligation-mediated polymerase chain reaction. The chromatin structure of the Xist promoter was found to be consistent with the activity state of the Xist gene. The silent allele (on the active X chromosome) showed no footprints, while the expressed allele (on the inactive X chromosome) showed footprints at a consensus sequence for a CCAAT box, two weak Sp1 sites, and a weak TATA box.

Preparation of spermatogonia, spermatocytes, and round spermatids for analysis of gene expression using fluorescence-activated cell sorting.

A new method is described for the purification of spermatogenic cell populations from mouse testis. Through use of this method, it is possible to purify leptotene, zygotene, and pachytene primary spermatocytes as well as round spermatids from adult mouse testis. In addition, spermatogonial populations can be purified from mice at 9 days postpartum. The leptotene and zygotene primary spermatocytes that can be prepared by this method are impossible to separate successfully by the unit gravity method. The cells were used to prepare RNA for reverse transcriptase-polymerase chain reactions.

Mouse endogenous X-linked genes do not show lineage-specific delayed inactivation during development.

X chromosome inactivation (XCI) has been assumed to be complete in all cells of female mouse embryos at about 6 d post coitum (dpc). However, a recent study on beta-galactosidase expression of an X-linked lacZ transgene suggests that XCI is probably not complete several days after this time in some lineages. To help resolve this issue, we analysed XCI in embryos which carry the T(X;16)16H (Searle's) translocation and are heterozygous at the X-linked Hprt and Pgk-1 genes. The quantitative RT-PCR single nucleotide primer extension (SNuPE) assay was used to measure Hprt and Pgk-1 allele-specific transcripts in embryos 9.5 dpc. No transcripts from the normal X chromosome were found in any of the tissues tested, indicating that inactivation was complete for these endogenous genes.

Quantitative RT-PCR assays show Xist RNA levels are low in mouse female adult tissue, embryos and embryoid bodies.

We have investigated expression of the Xist gene in mouse female adult kidney, embryos and embryonic stem (ES) cells undergoing in vitro differentiation as embryoid bodies. Using the quantitative RT-PCR single nucleotide primer extension (SNuPE) assay, we found that the amount of Xist RNA in adult kidney of three mouse strains was less than approximately 2000 transcripts per cell, with only modest differences between strains carrying different Xce alleles. Female embryos 7.5 days post coitum had the same number of Xist transcripts per cell as isogenic adult tissue. Using quantitative oligonucleotide hybridization assays after RT-PCR, we investigated Xist expression in ES lines heterozygous at the Pgk-1 and Xist loci. We found that, while in most (XX) ES lines Xist RNA levels increased during embryoid body formation, the levels seen were less than 10% those found in adult female kidney. In addition, we found that the allelic ratio of Xist transcripts from reciprocal (XX) ES cell lines differentiating in vitro was identical to that of isogenic 10.5 to 11.5 day female embryos. These latter results suggest that there is no pattern of preferential paternal imprinting during days 1 to 9 of in vitro differentiation of ES cells. However, the influence of the Xce locus on the randomness of X-inactivation in embryos seems to operate also in ES cell lines. Our overall conclusion is that the low levels of Xist RNA in female kidney, embryos and differentiating (XX) ES cells are compatible only with models that do not require Xist RNA to cover the entire inactive X chromosome.

Methylation analysis by genomic sequencing of 5' region of mouse Pgk-1 gene and a cautionary note concerning the method.

We have used genomic sequencing aided by ligation-mediated PCR (LMPCR) to assay for 5-methylcytosine in the CpG-rich promoter region of the mouse X-linked phosphoglycerate kinase gene (Pgk-1). Earlier studies showed that there was very heavy methylation of CpG dinucleotides in the CpG-rich promoter of the human PGK1 gene on the inactive X chromosome (the Xi), but that these same sites were completely unmethylated on the active X chromosome (the Xa). For mouse Pgk-1, previous restriction enzyme analysis had shown apparently complete methylation of only one cytosine in the promoter region on the Xi, at HpaII site H7, which is located in the untranslated region, 28 nucleotides upstream of the translation start site. We analyzed this potentially critical region by combining the use of HpaII with LMPCR, and find that the CpG dinucleotides near H7 are either unmethylated or only partially methylated on the Xi. LMPCR analysis of male and female DNA over a 490-bp sequence including the promoter and enhancer extend the finding of relative hypomethylation on the mouse Xi to include all CpG dinucleotides in this region. These results are relevant to the role of DNA methylation in stabilizing the inactive state of chromatin. In addition, we find that caution must be exercised in using LMPCR for methylation analysis of some sequences. A DNA concentration-dependent band-suppression artifact can incorrectly suggest methylation of both CpG and nonCpG dinucleotides.

Parental imprinting studied by allele-specific primer extension after PCR: paternal X chromosome-linked genes are transcribed prior to preferential paternal X chromosome inactivation.

The preferential inactivation of the paternal X chromosome in extraembryonic cells during early mouse development is an example of parental imprinting, but it has not been studied at the transcriptional level because standard methods of measuring RNA levels do not allow detection of allele-specific RNAs in individual early embryos. We sought to determine whether the paternal allele of the X chromosome-linked gene for 3-phosphoglycerate kinase 1 (Pgk-1), which is located very near the center of X chromosome inactivation, is transcribed prior to differentiation of extraembryonic lineages. Previous reports indicated that in heterozygous embryos there is a delay in the appearance of the phosphoglycerate kinase 1 allozyme encoded by the paternal X chromosome until 2 days after the appearance of the corresponding maternal allozyme. We report results obtained by use of a reverse transcription/PCR-based method which allows the quantitative measurement of allele-specific RNA. The assay is sensitive enough for the quantitative analysis in single embryos of allele-specific transcripts differing by only one nucleotide. We have used this assay to analyze mouse embryos heterozygous at the Pgk-1 and Hprt [hypoxanthine (guanine) phosphoribosyltransferase] loci, and we find that individual 8-cell and blastocyst embryos express both Hprt and Pgk-1 paternal transcripts, as do pooled 2- to 4-cell embryos. These results are discussed in view of the apparent temporal delay in paternal expression of the Pgk-1 gene at the enzyme level.

A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide.

We have found that the single nucleotide primer extension assay, a PCR-based assay currently used qualitatively to measure allelic differences in DNA, can be used quantitatively to measure allele-specific transcripts differing by only a single nucleotide. We show that total RNA containing the Pgk-1a transcript can be specifically detected even in a 1000-fold excess of RNA containing the Pgk-1b transcript.

A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.

Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation.

A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.