Isolating promoters of multigene family members from the polyploid sugarcane genome by PCR-based walking in BAC DNA

Genome ◽  
2010 ◽  
Vol 53 (10) ◽  
pp. 840-847 ◽  
Author(s):  
Mona B. Damaj ◽  
Phillip D. Beremand ◽  
Marco T. Buenrostro-Nava ◽  
John Ivy ◽  
Siva P. Kumpatla ◽  
...  
Keyword(s):  
Multigene Family ◽  
Family Members ◽  
Genome Walking ◽  
Cdna Sequences ◽  
Regulated Expression ◽  
Specific Amplification ◽  
Polyploid Genome ◽  
Polymerase Chain ◽  
Genomic Regions ◽  
Target Promoter

The availability of a wider range of promoters for regulated expression in valuable transgenic crops would benefit functional genomics studies and current biotechnology programs aimed at improved productivity. Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to isolate promoters or 5′ flanking genomic regions adjacent to known cDNA sequences in genomes that are not yet completely sequenced. However, these techniques are problematic when applied directly to DNA isolated from crops with highly complex and large genomes. An adaptor ligation-mediated PCR-based BAC genome walking method is described here for the efficient isolation of promoters of multigene family members, such as the putative defense and fiber biosynthesis DIRIGENT genes that are abundant in the stem of sugarcane, a species with a highly polyploid genome. The advantage of this method is the efficient and specific amplification of the target promoter using BAC genomic DNA as template for the adaptor ligation-mediated PCR walking.

scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

PCR BY THERMAL CONVECTION

2004 ◽  
Vol 18 (16) ◽  
pp. 775-784 ◽  
Author(s):  
DIETER BRAUN
Keyword(s):  
Polymerase Chain Reaction ◽  
Thermal Convection ◽  
Dna Amplification ◽  
Reaction Vessel ◽  
Cold Region ◽  
Chain Reaction ◽  
Heating And Cooling ◽  
Highly Sensitive ◽  
Specific Amplification ◽  
Polymerase Chain

The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method.

scholarly journals Identification of molecular defects in a subject with type I CD36 deficiency

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3545-3552 ◽  
Author(s):  
H Kashiwagi ◽  
Y Tomiyama ◽  
S Kosugi ◽  
M Shiraga ◽  
RH Lipsky ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Stop Codon ◽  
Type I ◽  
Molecular Defects ◽  
Cdna Sequences ◽  
Cd36 Deficiency ◽  
Translation Stop Codon ◽  
Polymerase Chain ◽  
Exon 4 ◽  
Splice Junctions

Abstract We performed a molecular analysis of a subject whose platelets and monocytes did not express any cell surface CD36 (designated as a type I CD36 deficiency). Amplification of the 5′ half of platelet and monocyte CD36cDNA (corresponding to nucleotide [nt] 191–1009 of the published CD36 cDNA sequence [Oquendo et al, Cell, 58:95, 1989]) showed that two different-sized CD36 cDNAs existed. One cDNA was of predicted normal size, whereas the other was about 150 bp smaller than that predicted for normal CD36 cDNA. Amplification of the 3′ region of CD36 cDNA (nt 962–1714) in this subject showed only normal-sized CD36 cDNA. Cloning and nt sequence analysis of the cDNAs showed that the smaller sized CD36 cDNA had 161-bp deletion (from nt 331 to 491), and a dinucleotide deletion starting at nt position 539. The same dinucleotide deletion was also detected in the normal sized CD36 cDNA. Both deletions caused a frameshift leading to the appearance of a translation stop codon. RNA blot analysis and quantitative assay using the reverse transcription- polymerase chain reaction (RT-PCR) showed that the CD36 transcripts in both platelets and monocytes were greatly reduced. Comparison of the determined cDNA sequences with the genomic DNA sequence for the human CD36 gene showed that the dinucleotide deletion was located in exon 5, and that the 161-bp deletion corresponded to a loss of exon 4. PCR- based analysis using genomic DNA showed that this subject was homozygous for the dinucleotide deletion in exon 5. Except for the dinucleotide deletion, we could not find any abnormalities around exon 3, 4, and 5 including the splice junctions. These results suggested that the deletions in CD36 mRNA were likely to be responsible for instability of the transcripts, and the dinucleotide deletion in exon 5 might affect the splicing of exon 4.

scholarly journals Qualitative Polymerase Chain Reaction Methods for Detecting Major Food Allergens (Peanut, Soybean, and Wheat) by Using Internal Transcribed Spacer Region

2009 ◽  
Vol 92 (5) ◽  
pp. 1464-1471 ◽  
Author(s):  
Takashi Hirao ◽  
Satoshi Watanabe ◽  
Yusuke Temmei ◽  
Masayuki Hiramoto ◽  
Hisanori Kato
Keyword(s):  
Internal Transcribed Spacer ◽  
Its Region ◽  
Detection Methods ◽  
Target Species ◽  
Internal Transcribed Spacer Region ◽  
Qualitative Polymerase Chain Reaction ◽  
Target Dna ◽  
Allergen Detection ◽  
Specific Amplification ◽  
Polymerase Chain

Abstract Allergen detection methods for peanut, soybean, and wheat were developed by designing PCR primer pairs for specific amplification of a fragment of the internal transcribed spacer (ITS) region reported for Arachis spp. for peanut, Glycine spp. for soybean, and Triticum and Aegilops spp. for wheat. The target species for detection included not only cultivated, but also wild and ancestor species, which were thought to be potentially allergenic. The ability of the resultant primer pairs to detect the target species was verified using genomic DNA extracted from A. hypogaea for peanut and G. max for soybean; T. aestivum, T. turgidum, T. durum, T. aestivum-rye amphidiploid, T. monococcum, T. timopheevi, Ae. speltoides, and Ae. squarrosa for wheat. The LODs were 50500 fg of target DNA, which were comparable to those of the most sensitive PCR methods previously reported. The results from the present work, as well as those from our previous work on buckwheat and kiwifruit, prove that the ITS region, for its high copy number and interspecific diversity, is particularly useful as the target of allergen detection methods.

Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR

2005 ◽  
Vol 56 (8) ◽  
pp. 1127 ◽  
Author(s):  
D. G. Bourne ◽  
R. L. Blakeley ◽  
P. Riddles ◽  
G. J. Jones
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
In Situ Hybridisation ◽  
Cyanobacterial Blooms ◽  
Chain Reaction ◽  
Fluorescent In Situ Hybridisation ◽  
Cyanobacterial Toxin ◽  
Specific Amplification ◽  
Polymerase Chain

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) techniques were developed for the detection of a Sphingomonas bacterium (strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA (rDNA) of strain MJ-PV. A 16S ribosomal RNA (rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.

Differentiation of Vibrio alginolyticus Strains Isolated from Sardinian Waters by Ribotyping and a New Rapid PCR Fingerprinting Method

1999 ◽  
Vol 65 (5) ◽  
pp. 1871-1875 ◽  
Author(s):  
Stefania Zanetti ◽  
Antonella Deriu ◽  
Ilaria Duprè ◽  
Maurizio Sanguinetti ◽  
Giovanni Fadda ◽  
...  
Keyword(s):  
Genetic Relationships ◽  
Restriction Enzymes ◽  
Epidemiological Studies ◽  
Vibrio Alginolyticus ◽  
Discriminative Power ◽  
Pcr Fingerprinting ◽  
Rapid Pcr ◽  
Specific Amplification ◽  
Genomic Regions ◽  
Potential Use

ABSTRACT We investigated the usefulness of a novel PCR fingerprinting technique, based on the specific amplification of genomic regions, to differentiate 30 Vibrio alginolyticus strains isolated in Sardinian waters. The different profiles obtained were scanned and analyzed by a computer program in order to determine genetic relationships. The results were then compared with the patterns obtained by ribotyping with HindIII, KpnI, andXbaI restriction enzymes. PCR fingerprinting could differentiate the strains analyzed into 12 different patterns, whereas ribotyping with XbaI, which produced the highest number of patterns, generated only 7 different profiles. This study revealed the superior discriminative power of the proposed technique for the differentiation of related V. alginolyticus strains and the potential use of PCR fingerprinting in epidemiological studies.

Rapid cycle allele-specific amplification: studies with the cystic fibrosis delta F508 locus

1993 ◽  
Vol 39 (5) ◽  
pp. 804-809 ◽  
Author(s):  
C T Wittwer ◽  
B C Marshall ◽  
G H Reed ◽  
J L Cherry
Keyword(s):  
Cystic Fibrosis ◽  
Dna Amplification ◽  
Temperature Cycling ◽  
Rapid Cycling ◽  
Cycle Times ◽  
Product Specificity ◽  
Annealing Temperatures ◽  
Allele Specific ◽  
Specific Amplification ◽  
Polymerase Chain

Abstract Rapid cycle DNA amplification is a polymerase chain reaction technique with improved product specificity and cycle times of 20-60 s, allowing complete 30-cycle reactions in 10-30 min. The presence or absence of the delta F508 deletion and wild-type allele was determined in 104 cystic fibrosis patients by rapid cycle DNA amplification. In separate allele-specific assays, sequences on both sides of the delta F508 locus were amplified with the 3' end of a discriminating primer at the delta F508 locus, with either a 3-bp or a 1-bp mismatch. With rapid cycling (35-s cycles), single-base discrimination was achieved over a broad range of annealing temperatures (50 degrees C or lower); with conventional cycling and "hot starts" (160-s cycles), only annealing temperatures of 61-62 degrees C sufficiently discriminated between alleles. With rapid cycling, genotype could still be assessed with annealing temperatures as low as 25 degrees C. We conclude that faster temperature cycling can improve the results of allele-specific amplification.

scholarly journals Detection of minimal residual disease in acute myelomonocytic leukemia with abnormal marrow eosinophils by nested polymerase chain reaction with allele specific amplification

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2291-2296 ◽  
Author(s):  
J Hebert ◽  
JM Cayuela ◽  
MT Daniel ◽  
R Berger ◽  
F Sigaux
Keyword(s):  
Residual Disease ◽  
Fusion Transcript ◽  
Type A ◽  
Chain Reaction ◽  
Allele Specific ◽  
Acute Myelomonocytic Leukemia ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Myelomonocytic Leukemia ◽  
Minimal Residual

Abstract Acute myelomonocytic leukemia with bone marrow eosinophilia (AML-M4Eo in the French-American-British FAB] classification) is frequently associated with pericentric inversion of chromosome 16, inv(16)(p13q22). Recently, the molecular cloning of teh breakpoints has led to the identification of the two fused genes, CBFB on 16q and MYH11 on 16p. We have analyzed 24 patients with AML-M4Eo at diagnosis and 47 patients with AML of other FAB subtypes, by a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the CBFB/MYH11 fusion mRNAs. Three types of fusion mRNAs were detected in 22 samples of AML- M4Eo (type A, n = 20; type C, n = 1; and type D, n = 1). Among these 22 positive samples, inv(16) was found in the 20 cytogenetically studied cases. No fusion transcript was detected in two patients with AML-M4Eo and in patients with other types of AML. These results confirm that CBFB/MYH11 transcripts (with a predominant type A form) are present in most cases of inv(16) AML. Moreover, detection of the hybrid transcript is closely associated with the finding of abnormal bone marrow (BM) eosinophils in AML-M4Eo as it is not found in other, FAB subtypes of AML, including AML-M4. To assess the presence of type A CBFB/MYH11 fusion transcripts in five AML-M4Eo patients in remission, we designed a sensitive assay combining nested PCR and allele-specific amplification (NPASA). Residual leukemia cells were detected in four patients who were in remission from 4 to 22 months, but not in one patient in long-term remission (5 years). The clinical relevance of persistent CBFB/MYH11 fusion transcripts in remission remains to be established by studying a large prospective series of patients. NPASA provides a useful and sensitive tool for the detection of minimal residual disease in inv(16) AML and, potentially, in other leukemias associated with translocations that result in a predominant fusion transcript.

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