Prenatal exclusion testing for Huntington disease using the polymerase chain reaction

Prenatal exclusion of Huntington disease (HD) may be carried out by analysis of cosegregating DNA markers on a first-trimester chorionic villus sample. The conventional Southern blot method is time-consuming and requires microgram quantities of DNA and milligram quantities of villus tissue. The use of the polymerase chain reaction (PCR) to amplify genomic DNA by a factor of 10(7) or more makes it possible to do analyses on very small samples in a few hours and without recourse to Southern blotting or hybridization with radioactive probes. We report on a fetus at risk of HD; prenatal testing was carried out by using the PCR to amplify a polymorphic DNA sequence adjacent to the HD locus. The risk of the fetus inheriting the HD gene could not be excluded and the pregnancy was terminated. This represents an example of gene tracking by using amplification of a restriction fragment length polymorphism at some distance from the relevant mutation.     

The gene for the iron sulfur protein of succinate dehydrogenase (SDH-IP) maps to human chromosome 1p35-36.1

A partial human cDNA clone for the iron-protein (IP) subunit of succinate dehydrogenase (EC 1.3.99.1) was used in Southern analyses of restriction enzyme digests of genomic human and hamster DNA as well as hamster-human hybrids containing a limited number of human chromosomes. The gene for this protein was mapped to human chromosome 1. Digestion of genomic DNA with several restriction enzymes yielded two fragments detectable on a Southern blot, in contrast to the expectations based on the sequence of the cDNA clone. A preliminary analysis of a genomic clone with most of theIP gene has indicated the presence of several introns containing restriction sites detected by the Southern analysis. This genomic clone was also used for subregional mapping by fluorescence in situ hybridization (FISH) to human metaphase chromosomes. A single locus in the region 1p35-36.1 was identified.     

Single DNA marker generated by "YAC-Alu PCR" that is end-specific.

A simple strategy for the rapid preparation of an end-specific linking-DNA probe from the YAC-human chromosome 21 DNA recombinant clone and the characterization of this single DNA probe are described. Synthetic oligodeoxynucleotide primers, based on the consensus Alu sequence, and the Sup4 DNA fragment in the YAC arms were used to amplify end-specific DNA sequences by the polymerase chain reaction (PCR) for screening of the linking YAC recombinant clones ("YAC-Alu PCR"). Nucleotide sequencing of the product of PCR from human genomic DNA in a YAC insert confirmed the boundary between the vector and the insert and the presence of the 3'-end Alu-like structure. The probe R1, prepared by "YAC-Alu PCR" amplification, was assigned to chromosome 21 by Southern hybridization of somatic cell hybrid DNAs. In situ hybridization allowed localization of the R1 DNA probe to the human chromosome 21q21-q22.1 region. Thus, this approach has significant advantages not only for isolation of a single DNA probe specific for human chromosome 21 but also for the screening of YAC linking recombinant clones for mapping of the human genome.     

Mapping of the P2U purinergic receptor gene to human chromosome 11q13.5–14.1

We mapped a human P2U purinergic receptor gene to chromosome 11q13.5–14.1. Oligonucleotide primers complementary to a part of the human P2U purinergic receptor cDNA were used to amplify a region from genomic DNAs from a panel of mouse/human somatic cell hybrid cell lines, each containing a single human chromosome. A PCR product of the expected size (378 bp) resulted from a single hybrid cell line containing human chromosome 11. The gene was further localized to a region of chromosome 11 using a sub-chromosomal hybrid panel containing different segments of chromosome 11. Based on the specific PCR product obtained and its Southern hibridization to the P2U receptor cDNA, the human P2U receptor gene was localized to chromosome 11q13.5–14.1.     

Analysis of region-specific library constructed by sequence-independent amplification of microdissected fragments surrounding weaver (wv) gene on mouse chromosome 16

The C3–C4 region of mouse chromosome 16 was microdissected and amplified directly by sequence-independent amplification (SIA). The SIA product was proved to originate from the microdissected region by fluorescence in situ hybridization (FISH) and was cloned into the PCR II vector (mean insert size 506 bp). Colony hybridization showed that about 59% of the clones contained either unique or low copy number sequences. Southern blot analysis of 100 unique clones demonstrated that 50 clones hybridized with single (33 clones) or multiple (17 clones) bands on blots of DNA from a hamster-mouse hybrid cell line that contains mouse chromosome 16, 13 clones hybridized with mouse but not with the hamster-mouse hybrid DNA, 19 clones contained repetitive sequences, and the remaining 18 clones failed to yield bands. One third of the 100 unique clones hybridized to human genomic DNA. Thirty-three clones were sequenced. None of them was found in GenBank. Our results demonstrate that this relatively simple method of microdissection and cloning can produce a library of good quality.     

Structural characterization and fine chromosomal mapping of the human P2Y1 purinergic receptor gene (P2RY1)

Using P2Y₁ specific oligonucleotide primers in a Polymerase Chain Reaction on human genomic DNA, we have amplified a region encoding the P2Y₁ receptor. Restriction analysis and Southern hybridization of the PCR product revealed that the entire open reading frame of the human P2Y₁ receptor is coded by an intronless gene. We have previously localized the P2Y₁ receptor gene to human chromosome 3. The gene was further localized to a region of chromosome 3 using a subchromosomal hybrid panel containing different segments of chromosome 3. Based on the specific PCR product obtained and its Southern hybridization to the human P2Y₁ receptor cRNA, the P2Y₁ receptor gene was mapped to human chromosome 3q25.     

Colocalization of P2Y2 and P2Y6 receptor genes at human chromosome 11q13.3-14.1

Extracellular nucleotides mediate a number of physiological responses through either ligand gated P2X or G protein-coupled P2Y receptors. To date, six P2Y receptor subtypes, P2Y1-P2Y6, have been cloned. We mapped the human P2Y6 receptor gene to chromosome 11q13.3-13.5. Oligonucleotide primers complementary to a part of the human P2Y6 receptor cDNA were used to amplify a region from genomic DNA from a panel of mouse/human somatic cell hybrid cell lines, each containing a single human chromosome. A PCR product of the expected size (714 bp) resulted from a single hybrid cell line containing human chromosome 11. The gene was further localized to a region of chromosome 11 using a subchromosomal hybrid panel containing different segments of chromosome 11. Based on the specific PCR product obtained and its Southern hybridization to the P2Y6 receptor cDNA, the human P2Y6 receptor gene was localized to chromosome 11q13.3-13.5. Previously, we have localized the P2Y2 receptor gene to human chromosome 11q13.5-14.1. This is the first report of the clustering of the P2 receptor genes. The clustering of these two P2Y receptor subtypes suggests a relatively recent expansion of the gene family by gene duplication.     

Genomic determination of the CR1 (CD35) density polymorphism on erythrocytes using polymerase chain reaction amplification and HindIII restriction enzyme digestion.

The density of CR1 (the C3b receptor, CD35) on erythrocytes from normal individuals is determined by a codominant bi-allelic system associated with a single base mutation within an intron of the CR1 structural gene, leading to an additional polymorphic HindIII endonuclease site. The CR1 genotype is determined by HindIII digestion of genomic DNA and Southern blotting. We have developed a method based on polymerase chain reaction (PCR) amplification of the genomic DNA fragment of interest followed by HindIII endonuclease digestion and agarose gel electrophoresis which permits a more rapid and reliable determination of the CR1 genotype. The method is suitable for large scale clinical studies in diseases with altered expression of CR1 on erythrocytes.     

Identification of a deletion in the mismatch repair gene,MSH2, using mouse-human cell hybrids monosomal for chromosome 2

Hereditary non-polyposis colorectal cancer is characterized by mutations in one of the DNA mismatch repair genes, primarily MLH1, MSH2, or MSH6. We report here the identification of a genomic deletion of approximately 11.4 kb encompassing the first two exons of the MSH2 gene in two generations of an Ohio family. By Southern blot analysis, using a cDNA probe spanning the first seven exons of MSH2, an alteration in each of three different enzyme digests (including a unique 13-kb band on HindIII digests) was observed, which suggested the presence of a large alteration in the 5' region of this gene. Mouse-human cell hybrids from a mutation carrier were then generated which contained a single copy each of human chromosome 2 on which the MSH2 gene resides. Southern blots on DNA from the cell hybrids demonstrated the same, unique 13-kb band from one MSH2 allele, as seen in the diploid DNA. DNA from this same monosomal cell hybrid failed to amplify in polymerase chain reactions (PCRs) using primers to exons 1 and 2, demonstrating the deletion of these sequences in one MSH2 allele, and the breakpoints involving Alu repeats were identified by PCR amplification and sequence analysis.     

Characterization of the mouse collectin gene locus.

Three of the four known mouse collectin genes have been mapped to chromosome 14. To further characterize the spatial relationship of these genes, a bacterial artificial chromosome (BAC) library of mouse chromosome 14 was screened using mouse surfactant protein (SP)-A and -D complementary DNAs (cDNAs). One large clone hybridized to both SP-A and SP-D cDNAs and was found by polymerase chain reaction (PCR) to contain sequences from one of the mouse mannose-binding lectin genes (Mbl1). We used Southern mapping and subcloning of overlapping restriction fragments to characterize the gene locus. Mapping was confirmed by fluorescent in situ hybridization of fiber-stretched BAC DNA and by Southern hybridization of restriction endonuclease-digested and PCR-amplified genomic DNA. We found that the SP-A, Mbl1, and SP-D genes reside contiguously within a 55-kb region. The SP-A and Mbl1 genes are in the same 5' to 3' orientation and 16 kb apart. The SP-D gene is in the opposite orientation to the two other collectin genes, 13 kb away from the 3' end of the Mbl1 gene. The mouse SP-D gene had not previously been characterized. We found its size (13 kb) and organization to be similar to that of human SP-D. Exon I is untranslated. The second exon is a hybrid exon that contains signal for initiation of translation, signal peptide, N-terminal domain, and the first seven collagen triplets of the collagen-like domain of the protein. Four short exons (III through VI) encode the collagen-like domain of the protein, and exons VII and VIII the linking and the carbohydrate-recognition domains, respectively.     

Interferons as gene activators: a cluster of six interferon-activatable genes is linked to the erythroid alpha-spectrin locus on murine chromosome 1

Several interferon-activatable murine genes were mapped to murine chromosomes by hybridizing cDNA probes to Southern blots of genomic DNA samples from a panel of mouse-hamster somatic cell hybrid lines. The 12 gene is located on chromosome 12 and it specifies a 3.6-kb mRNA. The 204 gene (specifying a 2.5-kb mRNA), and three genes of the 203 gene family (hybridizing to five mRNAs of sizes between 2 and 4.5 kb), together with the 202 gene (specifying a 2-kb mRNA) are located on murine chromosome 1. By restriction fragment length polymorphism analysis of DNA samples prepared from a panel of recombinant inbred mouse lines (C57BL/6J D DBA/2J) and from 85 [C3H/HeJ-gld/gld x Mus spretus) F1 X C3H/HeJ-gld/gld] backcross mice we established a close linkage of the 202, 203, and 204 genes to the erythroid alpha-spectrin gene (Spna-1) on distal murine chromosome 1. Cosmids containing the 202, 203, and 204 genes were isolated from a library derived from AKR mouse DNA. Southern blot analysis of such cosmids revealed: (a) hybridization of a partial 203 cDNA to three genes of the 203 gene family; (b) cross-hybridization of the 202 and 204 genes with one another and with a third gene (designated as 201 gene), and (c) a close linkage of genes of the 203 family with the 201, 202, and 204 genes. These results indicate the existence of a cluster of at least six closely linked, interferon-activatable genes on distal murine chromosome 1 in the vicinity of the Spna-1 locus and also of the Minor lymphocyte stimulating locus (Mlsa).     

Selective cloning of B cell hybridoma-specific rearranged immunoglobulin gene loci using the polymerase chain reaction

The use of conventional DNA cloning procedures to obtain productively rearranged Ig genes from B cell hybridomas for structure/function analysis of immunoglobulins is tedious and time-consuming. Here we describe a procedure based on PCR which permits rapid, selective isolation of DNA segments containing individual hybridoma-specific Ig gene rearrangements. The method, an adaptation of the so-called 'inverted PCR' technique (IPCR), can be applied most efficiently to specific genes where a preliminary restriction map is available from Southern blot analysis of the hybridoma genomic DNA. To achieve amplification of a given rearranged Ig locus, small amounts of total hybridoma DNA are digested to completion with a chosen restriction endonuclease and the fragments circularised by DNA ligase. Cleavage of the DNA circles using a second restriction enzyme, chosen specifically to cut 3' to a rearranged V-(D)-J exon, leads to linear DNA segments where the rearranged gene is now flanked by segments of known nucleotide sequence derived originally from the 3' region of the Ig H or L chain gene locus. This permits the selection of oligonucleotides that provide convergent primers for specific amplification of DNA segments containing the required gene rearrangement. Amplified DNA fragments can be cloned and rapidly characterised by sequence analysis.     

Mutation screening of bleomycin-induced V79 Chinese hamster hprt mutants using multiplex polymerase chain reaction.

We have shown by the filter hybridization technique that bleomycin (BLM) induces different types of mutations at the hprt gene locus of V79 Chinese hamster cells. DNA of mutants identified by Southern blots as partial deletions was subjected to further analysis using multiplex polymerase chain reaction (PCR) to localize the endpoints of the deletions over the hprt gene. The PCR analysis revealed that deletions occur in all parts of the hprt gene but are distributed non-randomly. Deletions occurred most frequently at the 3'-end of the hprt gene suggesting a possible existence of a hot spot for deletions in this region; exons 1, 2 and 3 appeared to be less affected by deletions. As PCR can detect microdeletions which are below the limit of resolution of Southern blot hybridization we analysed 25 HPRT- mutants with Southern wild-type pattern to distinguish between point mutations and small deletions. Of these HPRT- mutants, all except five showed PCR amplification products identical to that of V79 wild-type cells. These results are consistent with previous Southern analyses indicating that a large portion of BLM-induced HPRT- mutants are real point mutations. Five mutants, however, showed differences in fragment sizes of single PCR products or did not yield one single exon fragment and thus are probably the result of deletions which were not to be detected by Southern analyses.     

建立单管双向荧光PCR方法检测NQO1基因609C/T多态性

目的 建立单管双向荧光PCR方法快速测定人NAD(P)H:醌氧化还原酶1[NAD(P)H:quinone oxidoreductase 1,NQO1]基因609C/T多态性.方法 以人NQO1基因中的609C/T位点,设计双向引物,优化反应条件,应用SYBR GreenⅠ双向荧光PCR扩增191份人基因组DNA标本,并通过对产物进行熔解曲线分析,根据产物Tm值进行等位基因单核苷酸多态性分型.对其中62份标本用经典的PCR-限制性片段长度多态方法进行基因型分型,验证结果准确性.结果 62份样本的单管双向荧光PCR方法的基因型结果与PCR-限制性片段长度多态法分型结果符合率100%.191份样本中,纯合野生型(CC)占28%,杂合型(CT)占50%,纯合突变型(TT)占22%.结论 单管双向荧光PCR方法检测NQO1基因609C/T多态性,操作简便、反应过程快速,结果直观,敏感性、准确性和稳定性好,适用于临床样本检测及流行病学调查研究.     

建立Ig基因富集的DNA库进行2F7单克隆抗体基因克隆

用小鼠型抗人小细胞肺癌单克隆抗体的2F7杂交瘤细胞DNA,经限制性内切酶EcoRI或BamHI完全酶解,通过Southern blot,用小鼠Ig基因Cr_(2a)、V_(H)、J_(H)和E_(H)作探针,富集阳性DNA,同时用pSV_2neo质粒为载体与这些DNA重组,经筛选获得了一批杂交阳性的克隆。     

The gene for the α2 chain of the human fibrillar collagen type XI (COL11A2) assigned to the short arm of chromosome 6

A cosmid clone (CosHcol.11) containing the α2(XI) collagen gene ( COL11A2 ) has been isolated. The gene contains conserved DNA and amino-acid sequences characteristic of fibril forming collagen, which is in accordance with the classification of type XI collagen as a fibrillar collagen. The genomic clone containing the α2(XI) gene has been used as probe in the Southern blot analysis of DNA from a panel of human/hamster somatic cell hybrids containing different numbers and combinations of human chromosomes. Synteny analysis revealed that only chromosome 6 showed complete concordant segregation with C0L11A2. Furthermore, the gene was regionally mapped to the short arm of chromosome 6 by using a hybrid which contained only the long arm of the chromosome.     

Coding region structure of interleukin-8 gene of human lung giant cell carcinoma LU65C cells that produce LUCT/interleukin-8: homogeneity in interleukin-8 genes

A 1.9-kb fragment containing an interleukin-8 (IL-8) coding region was amplified by the polymerase chain reaction (PCR) from the genomic DNA of human lung giant cell carcinoma LU65C cells that produce LUCT/IL-8 with N-terminal sequence of AVLPR. The coding region was found to consist of 4 exons and 3 introns as identical as that of the gene of MDNCF/IL-8 lacking N-terminal AVLPR. PCR using genomic DNAs from human polymorphonuclear leukocytes and mononuclear cells also provided the same 1.9-kb fragment as that from LU65C genomic DNA. Thus, it seems likely that human cells possess IL-8 genes with the homogeneous coding region so that they may first produce the same mature protein with N-terminal AVLPR (= LUCT) which was then truncated.