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AmpliGrease: 'Hot start' PCR using petroleum jelly
... AmpliGrease hot-start LM-PCR is a modification of the manual protocol for the use of petroleum jelly as a barrier separating two aqueous components until heated 10 . The components are numbered in the order of addition to the tube using a liquid-handling robot. ...
... Hot-start protocols, which have proved so useful for optimizing PCR 8,9 , have remained largely unexplored for use with LM-PCR. One such method uses Ampligrease, which is petroleum jelly suspended in mineral oil, to separate two aqueous premix components until heated, whereupon the jelly becomes miscible with the oil, allowing the top premix to combine with the bottom 10 . In this report, we demonstrate that combining this hot-start method with the single-tube approach of Cairns and Murray 11 , as modified by us 3 , yields excellent results for Maxam-Gilbert sequencing (Fig. 1C). ...
Ligation-mediated polymerase chain reaction (LM-PCR) is a genomic analysis technique for determination of (1) primary DNA nucleotide sequences (2) cytosine methylation patterns (3) DNA lesion formation and repair, and (4) in vivo protein-DNA footprints. However, LM-PCR can be limited by the multiple steps required and the relatively short stretch of sequence (usually <200 bp) that can be analyzed per reaction. We report here a simplified, one-day LM-PCR protocol in which all pipetting steps can be performed by a robotic workstation and which, moreover, provides longer reads (>350 bp) and enhanced signal quality by use of nonradioactive detection and a LI-COR DNA sequencing instrument. Sensitivity comparable to radiolabeling is achieved using oligonucleotide primers that are 5'-end labeled with infrared fluorochromes. We showed that the technique could be used for sensitive and reproducible in vivo photofootprinting of the human phosphoglycerate kinase 1 (PGK1) promoter, as well as providing good Maxam-Gilbert sequence information. The methods described here should allow high-throughput, high-resolution analysis of transcription factor binding and chromatin structure, and also may be useful for sequencing gaps that are refractory to cloning.
... A 35 µ l reaction mixture contained 3.5 µ l of 10 × reaction buffer (750 mM Tris-HCl, pH 8.8, 200 mM ammonium sulfate, 0.1% Tween-20), 1 mM each dNTP, 12.5 pM each primer, 3.5 or 7 pM probe, 2.5 u Taq polymerase (DNK-Tekhnologiya), 5 µ l of cDNA solution, and 0.002 pg of a plasmid serving as internal control (IC). To reduce nonspecific hybridization by the hot start technique, the reaction mixture was prepared in two volumes, one containing the oligonucleotides, reaction buffer and dNTPs, and the other one containing cDNA and Taq polymerase, which were separated with a paraffin layer [28,18]. The PCR protocol optimized for a Terzik thermal cycler (DNK-Tekhnologiya) included 90 sec at 93°ë ; 5 cycles of 20 sec at 93°ë , 5 sec at 64°ë , and 5 sec at 67°ë ; and 40 cycles of 1 sec at 93°ë , 5 sec at 64°ë , and 5 sec at 67°ë . ...
Potato, one of the most widespread agricultural plants in Russia, is strongly affected by various pathogens of viral, bacterial, and fungal origin as well as by pests. Their simple and accurate diagnostics and identification sound rather important both for production of virus free planting material and to perform monitoring of the phytosanitary state of planting areas. Based on qualitative Fluorescent Amplification--based Specific Hybridization Polymerase Chain Reaction (FLASH-PCR) we have developed the diagnostic systems, which provided fast, careful, and with the minimum risk of contamination in the working zone by amplification products, detection of the major potato pathogens, i. e. A, Y, X, M, S potato viruses, potato leafroll virus, potato mop top virus, as well as potato spindle tuber viroids.
... There are several different methods for carrying out PCR hot start. These include: (i) the manual addition of the critical reaction components at the annealing temperature (1); (ii) the preliminary separation of the reaction components with a high-melting substance (2,3); (iii) the use of DNA polymerase antibodies (4); (iv) the use of chemically-modified AmpliTaq Gold DNA polymerase (PE Biosystems); (v) the use of oligonucleotides that reversibly inhibit DNA polymerase (5); and (vi) the use of thermolabile DNA helicases (6). ...
A new technique of PCR hot start using oligonucleotide primers with a stem–loop structure is developed here. The molecular
beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3′-end sequence of the primers
was complementary to the target and five or six nucleotides complementary to the 3′-end were added to the 5′-end. During preparation
of the reaction mixture and initial heating, the oligonucleotide has a stem–loop structure and cannot serve as an effective
primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension
can begin.
... Polymerase chain reaction is one of the main methods used in current molecular biology. Several approaches are now used to enhance its specificity: optimization of PCR conditions [1][2][3], hot start [4][5][6][7][8] and nested PCR [9,10], and the addition to the reaction mixture of compounds favoring the DNA melting [11][12][13][14][15][16]. ...
A new approach to enhanced specificity and product yield of polymerase chain reaction is proposed. It is based on control of DNA polymerase activity during PCR by changing the magnesium ion concentration, which depends on the temperature of the reaction mixture. A slightly soluble magnesium salt, magnesium oxalate, whose solubility depends on temperature, was used as a source of magnesium ions. During PCR, magnesium oxalate was maintained at saturating concentration by the presence of an insoluble excess of this salt, and the concentration of magnesium ions depended on the salt solubility: binding of magnesium ions at lower temperatures and their release at higher temperatures was shown to affect the DNA polymerase activity and to favor the specific PCR amplification of the target DNA fragment.
... This was followed by heat treatment (95°C) for 5 minutes to inactivate the MMLV- RT enzyme. The cDNA produced by the method described above was then amplified by polymerase chain reaction (PCR) using the ''hot-start'' method [45] in a 50 lL reaction volume, containing a mix of dNTP (0.2 mM of each), specific primers (0.5 mM of each), Taq DNA polymerase (1 unit), 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 1.5 mM MgCl 2 . RT-PCR primers used in the studies were as follows: b 2 microglobulin (237 bp), forward 5¢-ggAgACAgCACTCAAAg TAg and reverse 5¢-ggTgTgAACCATgTgACTTT; collagen type 1a(I) chain (235 bp), forward 5¢-CTTggTCTCgTCACA- gATC and reverse 5¢-CTTTTAACgTTggAggATgTgC- TATTTggC; LPL (324 bp), forward 5¢-gAAAgTgTCTAC TTTgCAgAAAggAAAAggC and reverse 5¢-CTTTTAACgTT ggAggATgTgCTATTTggC; G3PDH (568 bp), forward 5¢-TTg CAACTgTTTTAggACTTT and reverse 5¢-AgCATTgggAAA TgTTCAAgg; peroxisome proliferator-activated receptor (PPAR) c2 (310 bp), forward 5¢-TgAAACTCTgggAgATTCT CCTATTgACCC and reverse 5¢-TCAgAATAATAAggTgg AgATgCAggCTCC. ...
A consistent observation in osteoporosis is bone volume reduction accompanied by increased marrow adipose tissue. No single
cause linking the two phenomena has yet been identified. In a human progenitor cell clone (hOP 7) derived from bone marrow,
however, we have demonstrated that rabbit serum can direct differentiation away from an osteoblast lineage to one of adipocytes.
We now report on whether human serum has a similar effect. Serum was collected from 10 pre- and 10 postmenopausal women and
from the 10 postmenopausal women before and following 6-week hormone replacement therapy (HRT). hOP 7 cells were cultured
with the various sera, and after 7-14 days adipocytogenesis was determined by oil red O staining and lipoprotein lipase (LPL)
and glycerol 3-phosphate dehydrogenase (G3PDH) expression. Incubation with 10% premenopausal serum led to labeling of 10.9%
of cells (P<0.05) with oil red O, whereas application of 10% postmenopausal serum led to a much larger effect, 43.5% labeling (P<0.001 with respect to premenopausal serum). Oil red O positivity was accompanied by loss of type I collagen expression
and increased LPL and G3PDH expression. HRT did not reverse the adipocytogenic effect of postmenopausal serum. In conclusion,
serum from postmenopausal women contains factors that steer hOP 7 bone progenitor cells toward an adipocytic phenotype, irrespective
of HRT. The study suggests a role for serum factors in the development of fatty marrow in postmenopausal osteoporosis.
... A 35 µ l reaction mixture contained 3.5 µ l of 10 × reaction buffer (750 mM Tris-HCl, pH 8.8, 200 mM ammonium sulfate, 0.1% Tween-20), 1 mM each dNTP, 12.5 pM each primer, 3.5 or 7 pM probe, 2.5 u Taq polymerase (DNK-Tekhnologiya), 5 µ l of cDNA solution, and 0.002 pg of a plasmid serving as internal control (IC). To reduce nonspecific hybridization by the hot start technique, the reaction mixture was prepared in two volumes, one containing the oligonucleotides, reaction buffer and dNTPs, and the other one containing cDNA and Taq polymerase, which were separated with a paraffin layer [28,18]. The PCR protocol optimized for a Terzik thermal cycler (DNK-Tekhnologiya) included 90 sec at 93°ë ; 5 cycles of 20 sec at 93°ë , 5 sec at 64°ë , and 5 sec at 67°ë ; and 40 cycles of 1 sec at 93°ë , 5 sec at 64°ë , and 5 sec at 67°ë . ...
Potato is commonly affected by various pathogens of viral, bacterial, and fungal origin; therefore, simple and accurate diagnostic
and identification techniques are of key importance both for the production of virus free planting material and for the monitoring
of the phytosanitary state of planting areas. The newly developed test systems based on qualitative fluorescent amplification-based
specific hybridization (FLASH-PCR) enable fast and accurate diagnostics of the major potato pathogens, i.e. potato viruses
A, Y, X, M, and S, potato leaf roll virus, potato mop top virus, as well as potato spindle tuber viroid, while minimizing
the risk of the working zone contamination.
... Hot-start PCR has been implemented so far by mechanically separating a reagent (7), by blocking the DNA polymerase with an antibody (8) or by using AmpliTaq Gold ® (PE Biosystems, Foster City, CA, USA), a modified form of AmpliTaq DNA polymerase that is inactive at room temperature (1). In two recent reports, single-stranded DNA aptamers (oligonucleotides selected by an in vitro selection procedure) were shown to inhibit the activity of various DNA polymerases. ...
A new method to produce hot-start conditions in PCR is described. Short double-stranded DNA fragments were found to inhibit the activity of DNA polymerases from Thermus aquaticus and Thermus flavus. This inhibition is not sequence specific, but exclusively dependent on the melting temperature of the fragments as shown by its correlation to their melting curves as measured. This property is exploited by adding fragments of the appropriate length to the PCR mixture during the reaction setup and thereby preventing the DNA polymerases from extending primers annealed nonspecifically at lower than the optimal temperature. By amplifying ten copies of phage lambda DNA in the presence of 2 micrograms of nonspecific DNA, it is shown for three different primer pairs how the melting temperatures of the double-stranded DNA fragments have to be adapted to the cycle profiles to obtain predominantly specific products in the 0.5 microgram range.
... There are several different methods for carrying out PCR hot start. These include: (i) the manual addition of the critical reaction components at the annealing temperature (1); (ii) the preliminary separation of the reaction components with a high-melting substance (2,3); (iii) the use of DNA polymerase antibodies (4); (iv) the use of chemically-modified AmpliTaq Gold DNA polymerase (PE Biosystems); (v) the use of oligonucleotides that reversibly inhibit DNA polymerase (5); and (vi) the use of thermolabile DNA helicases (6). ...
A new technique of PCR hot start using oligonucleotide primers with a stem-loop structure is developed here. The molecular beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3'-end sequence of the primers was complementary to the target and five or six nucleotides complementary to the 3'-end were added to the 5'-end. During preparation of the reaction mixture and initial heating, the oligonucleotide has a stem-loop structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension can begin.
... Robotic LMPCR, which consists of three cascading stages of AmpliGrease (11), was carried out on a Biomek ® 2000 workstation (Beckman Coulter, Fullerton, CA, USA) as previously described (4) unless otherwise stated. For example, the dNTP concentration was changed to 800 µM for the primer extension step when processing UV-treated and DMS/AlkA-treated DNA. ...
The investigation of in vivo DNA repair in mammalian cells at nucleotide resolution requires the quantification of break frequencies less than one per kilobase. By optimizing several parameters of the ligation-mediated PCR technique, we find that the required sensitivity can be achieved. We also report details of a one-day procedure that can be performed either with or without a robotic liquid handling workstation. The use of near-infrared fluorescent-labeled primers with detection by a LI-COR DNA sequencer provides for safe, nonradioactive detection, similar in sensitivity to the use of 32P-labeled primers but with the additional advantage that high-quality digitized data are obtained directly. Multiplexing can be performed; that is, more than one sequence can be analyzed in a single reaction, and multiple reactions can be processed robotically. Primer sets for exons 5-8 of the tumor suppressor gene, p53, were designed for simultaneous thermal cycling. The improved procedure with infrared detection was used to monitor low-frequency damage (<1 break/kb) and/or repair of UVB, UVC, and chemical methylation. Quantitative data on the linearity of response and reproducibility are described. The coefficient of variation for technical replicates was typically 10%. The methods described here will permit high sample throughput for the detection of DNA damage and repair as well as in vivo protein footprints.
... Besides being tedious and prone to error, this manual method is very subject to contamination and cross-contamination of PCR samples. We use this method as a control, i.e. in Figures 2C (lanes 3 and 4), 3 ...
... Besides being tedious and prone to error, this manual method is very subject to contamination and cross-contamination of PCR samples. We use this method as a control, i.e. in Figures 2C (lanes 3 and 4), 3 ...
Although the thermophilic bacterium Thermus aquaticus grows optimally at 70°C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20–37°C.
This activity is a bane to some PCRs, since it catalyzes non‐specific priming. We report mutations of Klentaq (an N‐terminal
deletion variant) DNA polymerase that have markedly reduced activity at 37°C yet retain apparently normal activity at 68°C
and resistance at 95°C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near
the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation.
We show that the novel cold‐sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.
Polymerase chain reaction (PCR) now provides the basis for many molecular tests used in the clinical microbiology laboratory. PCR amplifies sequences of nucleic acids by using primers directed to the sequence of interest. Amplicon is generated by repeated cycles of denaturation, annealing, and extension. Variations on PCR also allow for quantification of nucleic acids, detection of multiple targets, and organism typing.
The propensity of Taq polymerase to add 3′-A overhangs (1,2) to PCR-amplified DNA has made possible a simple method for cloning PCR products into a T-vector (Invitrogen [San Diego, CA]; 3–5). Here we present a related strategy that uses T-linkers to add sequences, such as restriction sites, to the ends of PCR products (see Note 1). A single base T overhang at the end of a synthetic double-stranded oligonucleotide linker allows ligation of the linker to the unpolished ends of a PCR product. This avoids the expense of adding the “extra” sequences to sequence-specific primers.
So einfach die Durchführung einer PCR erscheint, so kompliziert kann die Fehlersuche sein, wenn das gewünschte Amplifikat nicht erhalten wird oder unspezifische Fragmente auftreten. Häufig auftretende „Fehler“ sind durch eine Fehlpaarung der Oligonucleotide charakterisiert. Diese Moleküle binden entweder an unspezifischen Stellen oder können auch als sogenannte Primer-Dimere miteinander interagieren. Generell sollten die Oligonucleotide mithilfe eines geeigneten Computer-Programms konstruiert werden, um eine optimale Bindung an die DNA-Matrize zu erreichen. Sofern die optimalen Oligonucleotide sowie PCR-Parameter für die Amplifikation eingesetzt werden, wird es zu keinem Auftreten unspezifischer Amplifikate kommen. Allerdings ist nicht immer vorhersehbar, welche ähnlichen Bindungssequenzen in der gesamten DNA-Matrize (z. B. genomische DNA) vorkommen. Im Falle der Primer-Dimere kann durch Einsatz der entsprechenden Software diese Bindung komplementärer Basen vorhergesehen und eliminiert werden.
Die PCR lässt sich auf vielen verschiedenen Wegen optimieren, wobei es keine allgemeingültigen Richtlinien für die Optimierung einer PCR gibt. Leider müssen für jedes nichtoptimale PCR-System die geeigneten Parameter gefunden werden, aber in den nachfolgenden Unterkapiteln sind etablierte Verfahren zum Gelingen einer effektiven PCR dargestellt.
Kary Mullis devised a method of replicating genes called "PCR" (polymerase chain reaction). A DNA sequence less than one part in a million of the total sample can be cloned. In fact, a single gene can be amplified into millions of duplicate copies. In order to determine the exact DNA sequence of a gene or section of DNA, it is necessary to have an adequate sample of the particular gene to work with. PCR allows a researcher to replicate a gene into a workable amount. PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR has become commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or environment. However, PCR has evolved far beyond simple amplification and detection, and many extensions of the original PCR method have been described.
The polymerase chain reaction (PCR) is an in vitro technique used to replicate, or amplify, a specific region of DNA billions-fold in a few hours or less [1–3]. The amplification is primer directed; oligonucleotide primers anneal to and flank the DNA region to be amplified. PCR is utilized in diagnostic and research laboratories to generate sufficient quantities of DNA to be adequately tested, analyzed, or manipulated. Because of the exquisite sensitivity it offers, PCR has rapidly become a standard method in diagnostic microbiology. More recently, reagent kits and various instrument platforms have added speed, flexibility, and simplicity [4–10]. How significant is the contribution of PCR to the field of biomedicine? A PubMed search in 2005 for the first edition of this book using the key word “PCR” produced 214,352 hits [11]. A search conducted in June 2011 using the same key word produced 259,042 hits.
Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30˚C and 56˚C. DNA targets were well amplified even after PCR mix-ture was pre-incubated 3 hr at 30˚C or 1 hr at 50˚C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentra-tion, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not pri-marily due to the reduced polymerase concentration resulted from the above interaction. This study pro-vided another good start to investigate potential im-plications of quantum dots in key molecular biology techniques.
Acetylcholine is synthesized and released by human epidermal keratinocytes and modulates the adhesion and motility of these cells, To understand the molecular basis of the effects of acetylcholine on keratinocytes, we investigated the presence, pharmacology, structure, and function of nicotinic acetylcholine receptors in human epidermal keratinocytes. Patch-clamp studies indicated that keratinocytes express acetylcholine receptors with ion gating and pharmacologic properties similar to those observed so far only in neurons, and containing the 3 subunit. Specific binding of the receptor-specific ligand 125I--bungarotoxin revealed approximately 5500 binding sites per cell on undifferentiated keratinocytes in cell cultures and approximately 35,400 binding sites per cell on mature keratinocytes freshly isolated from human neonatal foreskins, Antibody binding and polymerase chain reaction experiments demonstrated the presence of 3, 2, and 4 nicotinic receptor subunits. Binding of subunit-specific antibodies indicated that nicotinic receptors were associated with the suprabasal keratinocytes in epidermis and localized to the cell membranes of differentiated keratinocytes in cell cultures. Acetylcholine and the nicotinic agonist nicotine increased cell-substrate and cell-cell adherence of cultured keratinocytes and stimulated their lateral migration. The specific antagonists -bungarotoxin and mecamylamine caused cell detachment and abolished migration. Thus, a nicotinic receptor expressed in keratinocytes may mediate acetylcholine control of keratinocyte adhesion and motility.Keywords: radioligand binding, immuofluorescence, polymerase chain reaction, patch clamp
The discovery of restriction endonucleases is perhaps the most significant technical milestone in the development of recombinant DNA technology. For, together with ligation and transformation procedures, they enabled the insertion of genes of any organism in suitable vectors (e.g. plasmids), introduction of the vector in bacteria and subsequent replication of the DNA of interest during cell division (cloning). Other important technical milestones include the development of methods for DNA sequencing and the chemical synthesis of oligodeoxynucleotides.
The polymerase chain reaction (PCR) is another technique which had a major impact in every area of research that involves nucleic acid analysis. PCR is a method of exponentially increasing the concentration of a specific nucleic acid sequence in a reaction mixture. A great advantage of PCR is that the target nucleic acid does not need to be particularly pure. It can be a minute part of an extremely complex mixture of biological material. Crude cell lysates provide a very good starting material. The ability to propagate, in a few hours, minute amounts of DNA that are too small for standard amplification (i.e. cloning) techniques gives PCR an extraordinary power and sensitivity.
The advent of PCR stimulated the research activity in the development of alternative exponential amplification systems such as the ligase chain reaction (LCR), the self‐sustained sequence replication (3SR), the nucleic acid sequence‐based amplification (NASBA) and the strand displacement amplification system (SDA). An essential feature of target amplification techniques is that the amplification is an iterative process consisting of successive cycles. The products synthesized in one cycle act as templates for the next cycle.
Amplification techniques can be used to amplify trace amounts of nucleic acid sequences in blood, cells, water, food and other clinical and environmental samples. Fixed tissue specimens, buccal cells from mouth washes, human hair, dried blood at the scene of a crime, single lymphoid or sperm cells are examples of starting material that may be used for nucleic acid analysis.
Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagents, available on the Internet. This month's column discusses the failure of PCR primers that have been kept a long time to amplify DNA. For details on how partake in the newsgroup, see the accompanying box.
Molecular diagnostics has become an important part of the clinical laboratory, with tests and methods to identify a disease and understand the predisposition for a disease by analyzing DNA or RNA. Molecular diagnostics offers a growth opportunity in utilizing molecular tools to precisely target therapeutics and the development of biochips, micro fluidics technology and nanotechnology extends the limits of detection.
Molecular Diagnostics can provide gene profile based personalized therapeutic approaches, and therapy based on this molecular diagnostic information delivers effective treatment with the least toxicity. In the future, the scope of molecular diagnostics in molecular medicine could be expanded well beyond current nucleic acid testing, and it will play an important role in medicine, public health, pharmaceutics, forensics and drug discovery.
‘Molecular diagnostics: promises and possibilities’ covers:
• The identification of viable technology drivers through a comprehensive look at platform technologies for molecular diagnostics, including probe-based nucleic acid assays, microarrays and sequencing
• The most important molecular diagnostics tests: predictive, screening, prognostic, monitoring, pharmacogenomic and theranostic from their basic principles to their applications
• The discovery of feasible market opportunities by identifying high-growth applications in different clinical diagnostic areas and by focusing on expanding markets, such as communicable diseases, cardiology and oncology
• Global industry development through an in-depth analysis of the major world markets for moleculardiagnostics, including growth forecasts.
This much needed book is written for students, scientists and professionals working in the field of molecular diagnostics, as well as pathologists, medical microbiologists, pharmaceutical scientists, agricultural scientists and veterinary doctors. No other book currently available provides such a complete picture of the developments in the field of molecular diagnostics.
The polymerase chain reaction (PCR) is an in vitro technique used to replicate, or amplify, a specific region of DNA billions-fold in just a few hours (Saiki et al., 1985,
1988; Mullis and Faloona, 1987). The amplification is primer directed— oligonucleotide primers anneal to and flank the DNA
region to be amplified. PCR is used in diagnostic and research laboratories to generate sufficient quantities of DNA to be
adequately tested, analyzed, or manipulated. Because of the exquisite sensitivity it offers, PCR has rapidly become a standard
method in diagnostic microbiology. More recently, reagent kits and various instrument platforms have added speed, flexibility,
and simplicity (Tang et al., 1997; Fredricks and Relman, 1999; Tang and Persing, 1999). How significant is the contribution
of PCR to the field of biomedicine? This question is perhaps best answered by the results of a PubMed search using the key
word “PCR” (214,352 hits) or a search using the key words “PCR” and “diagnosis” (74,447 hits).
Control of gene transcription, the process in which a gene's DNA sequence serves as a template for mRNA synthesis, plays a critical role in the multistep process that regulates gene expression. Gene transcrtption levels within a cell change in response to a wide variety of signals that occur during cell development, differentiation, and normal physiological function. Changes in transcrip tion levels also occur in response to disease and other factors. In turn, these changes in transcription levels cause variations in the steady-state levels of indivtdual mRNAs. Thus, analysis of specific mRNA levels 1s vital in a broad range of research areas.
The polymerase chain reaction (PCR) has revolutionized both the basic and the applied aspects of the biomedical field with more than 40,000 papers having been published employing this technique. PCR has become an indispensable tool for basic research applications, such as cloning (1), sequencing (2), mRNA analysis (3), DNA and RNA quantitation (4-6), mutagenesis (7), and gene detection (8). Because of its ability to amplify minute quantities of nucleic acid specifically, PCR has also been applied with great success in clinical diagnostics (9-11), genetic testing (12-15), forensics (16,17), and environmental testing (18,19).
Hintergrund: Ziel dieser Studie war es, eine mögliche Beziehung zwischen vier Polymorphismen des TGF-beta1 Gens (-800G>A; -509C>T; Leu10Pro; Arg25Pro) und der Ausprägung, der Aktivität und der Progression (METAVIR-Score) einer Leberfibrose darzustellen. Methode: Drei Kollektive, 48 Patienten mit Leberfibrose (26 mit bekannter Hepatitis-C Virusinfektion), 47 Patienten mit nicht fibrosierenden Krankheiten und 50 gesunde Blutspender, wurden auf TGF-beta1 Polymorphismen mittels ARMS-PCR und Sequenzierungsanalysen untersucht. Die Konzentrationen von totalem TGF-beta1 wurden im Plasma und die von Hyaluronan, P?III-NP und der Transaminasen im Serum gemessen. Ergebnis: Das Vorhandensein von Prolin in Codon 10 und/ oder 25 war mit einer schnelleren Progression der Leberfibrose assoziiert. Patienten mit dem Genotyp 25ArgPro entwickeln signifikant schneller eine Fibrose (0,23 Units/ Jahr), als jene mit 25ArgArg (0,08 Units/ Jahr). Desweiteren war die Fibrose-Progression bei Patienten mit den Genotypen 10LeuPro und 10ProPro drei mal schneller als jene mit 10LeuLeu. Die Ausprägung und die histologische Aktivität der Fibrose war bei 25ArgPro im Vergleich zu 25ArgArg höher. Auch zeigte sich bei 10LeuPro eine stärkere Ausprägung als bei 10LeuLeu. Zwischen dem 25ArgArg und 25ArgPro Genotyp der Patienten mit Fibrose, zeigten sich keine signifikanten Unterschiede der TGF-beta1 Plasmakonzentrationen. Die Häufigkeit des 25ArgPro Genotyps bei den Leberfibrose-Patienten betrug das dreifache gegenüber der Kontrollgruppe, wohingegen die Häufigkeitsverteilung des 25ArgArg Genotyps innerhalb der Fibrose-Gruppe zu niedrigeren Werten tendierte. Die TGF-beta1-Promoter Polymorphismen zeigten keine Korrelation mit der Ausprägung, der entzündlichen Aktivität oder der Progression einer Leberfibrose. Schlussfolgerung: Die Ergebnisse belegen, daß der heterozygote Genotyp ArgPro des Codons 25 eine signifikant schnelle Fibrosierung der Leber bei Hepatitis-C Virus infizierten Patienten anzeigt, als 25ArgArg. Der homozygote LeuLeu Genotyp des Codons 10 zeigt eine langsamere Progression an. Background: The objective of the present study was to elucidate possible relationships between four polymorphisms of the TGF-beta1 gene (-800G>A; -509C>T; Leu10Pro; Arg25Pro) and stage, histological activity grade and progression rate (METAVIR-score) of liver fibrosis. Methods: Three study groups, i.e. 48 patients with hepatic fibrosis (26 with known duration of hepatitis C virus infection), 47 patients with non-fibrotic diseases and 50 healthy blood donors, were analyzed for TGF-beta1 polymorphisms using ARMS-PCR and sequence analysis. The concentrations of total TGF-beta1 in plasma and of hyaluronan, P-III-NP and activities of transminases in serum were measured. Results: The presence of proline at codons 10 and/ or 25 was associated with a faster progression of fibrosis than other polymorphisms. Patients with the genotype 25ArgPro developed fibrosis significantly faster (0.23 units/ year) than those having 25ArgArg (0.08 units/ year). Similarly, the fibrosis progression rate of patients with genotypes 10LeuPro and 10ProPro was almost three times as fast as of those having genotype 10LeuLeu. Stage and histological activity grade of fibrosis in 25ArgPro in comparison to 25ArgArg were higher. Also 10LeuPro showed a higher average stage of fibrosis than 10LeuLeu. The TGF-1 plasma concentrations of patients with hepatic fibrosis were not significantly different between carriers of 25ArgArg and 25ArgPro genotypes. The frequency of the genotype 25ArgPro in liver fibrotic patients was about three times that of the control group whereas the frequency distribution of the genotype 25ArgArg tended to lower frequency in the fibrosis group. TGF-beta1-promoter polymorphisms did not show any correlation with stage, grade or progression of liver fibrosis. Conclusion: The results indicate that the heterozygous ArgPro of codon 25 predicts significantly faster fibrotic progression of chronic hepatitis C than the homozygous 25ArgArg genotype. The homozygous LeuLeu genotype of codon 10 showed a slow progression of fibrosis.
2'-Deoxyribonucleoside-3'-O-(4-oxotetradec-1-yl) phosphoramidites (OXT phosphoramidites) are used to prepare modified oligodeoxyribonucleotide primers containing heat cleavable OXT phosphotriester protecting groups at 3'-ultimate and penultimate internucleotide linkages. The OXT-modified primers significantly improve performance of the polymerase chain reaction (PCR) compared to standard DNA primers by substantially reducing or eliminating the accumulation of PCR artifacts such as dimerized primers and misprimed amplicons. Basic protocols for synthesis of OXT-modified oligonucleotide primers and for performing hot-start PCR are described.
A new approach that improves the efficiency and specificity of polymerase chain reaction (PCR) has been developed. Heat-sensitive 3'-protected derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) have been synthesized and used as substitutes for natural dTTP, dCTP, dATP, and dGTP in PCR. Since 3'-protected dNTPs are either nonsubstrates or terminating substrates for Taq DNA polymerase, they do not support primer extension/elongation at low stringency conditions during PCR sample preparation when PCR artifacts such as primer dimers and mispriming products can form. At the initial heat-denaturing step and during the PCR sequence, the 3'-protecting group is cleaved, releasing 3'-unprotected dNTP that is a natural substrate for DNA polymerase. As a result, the primer extension/elongation proceeds only at an elevated temperature of PCR, when the interaction of primers and template is highly stringent and specific. Several 3'-protecting groups covering a wide range of deprotection kinetics have been tested. The 3'-O-tetrahydrofuranyl derivatives of dNTPs have demonstrated the best properties leading to a drastically reduced accumulation of PCR artifacts, such as "primer dimers" and "mispriming" products. Overall, PCR with 3'-THF-protected dNTPs demonstrated substantially improved performance and was more efficient and specific compared to PCR with standard dNTPs.
A novel means of recording the expression status of the total gene population is described. Digestion of cDNA by class IIS
restriction enzymes produces a fragment with a poly (A) stretch and a 5′ overhang with an unknown sequence. This fragment
contains information such as the class IIS enzyme that cuts cDNA nearest to the poly (A) stretch, the sequence of the 5′ overhang,
and the size of the fragment. Expressed genes can be discriminated and displayed by the fragment as follows: (i) cut the cDNA
with one class IIS restriction enzyme; (ii) ligate the digested cDNA to that from a pool of 64 biotinylated adaptors cohesive
to all possible overhangs; (iii) digest by other two class IIS enzymes; (iv) recover the ligated molecules with streptavidin-coated
paramagnetic beads; (v) perform PCR with the adaptor-primer and an anchored oligo-dT primer (vi) separate the amplified fragments
by denaturing polyacrylamide gel electrophoresis. Repeat the experiment with 64 adaptors, three enzymes and three anchored
oligo-dT primers displays most of the expressed genes. Because redundancy is minimized, this technique is also ideal for generating
tags for expressed genes, with which to construct a transcript map of the genome.
Human epidermal keratinocytes synthesize, secrete, and degrade acetylcholine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetylcholine. Because acetylcholine modulates transmembrane Ca2+ transport and intracellular metabolism in several types of cells, we hypothesized that cholinergic agents might have similar effects on keratinocytes. Nicotine increased in a concentration-dependent manner the amount of 45Ca2+ taken up by keratinocytes isolated from human neonatal foreskins. This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinocyte nicotinic acetylcholine receptor(s). The sequences encoding the α5 and α7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes. These subunits, as well as the α3, β2, and β4 subunits previously found in keratinocytes, can be components of Ca2+-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the race of cell differentiation, we measured the nicotinic cholinergic effects on the expression of differentiation markers by cultured keratinocytes. Long-term incubations with micromolar concentrations of nicotine markedly increased the number of cells forming cornified envelopes and the number of cells staining with antibodies to suprabasal keratin 10, transglutaminase type I, involucrin, and filaggrin. The increased production of these differentiation-associated proteins was verified by Western blotting. Because nicotinic cholinergic stimulation causes transmembrane Ca2+ transport into keratinocytes, and because changes in concentrations of intracellular Ca2+ are known to alter various keratinocyte functions, including differentiation, the subcellular mechanisms mediating the autocrine and paracrine actions of epidermal acetylcholine on keratinocytes map involve Ca2+ as a second messenger.
A random sequence library of single stranded DNA was screened to isolate sequences with high affinity for Thermus aquaticus DNA polymerase (Taq pol), a thermostable enzyme commonly used in the polymerase chain reaction (PCR). Selected oligonucleotide sequences bound Taq pol with dissociation constants in the low picomolar range, and efficiently inhibited polymerase activity at room temperature (20 to 25 degrees C), but did not inhibit at temperatures above 40 degrees C. Moreover, inhibition was thermally reversible. A process called "hot start" PCR is commonly used to prevent non-specific PCR products in amplification of low copy number targets. We show that the addition of oligonucleotide inhibitors eliminated the need for "hot start" conditions and improved the efficiency of detection of a low copy number target in PCR.
The polymerase chain reaction (PCR) with polyacrylamide gel electrophoresis was used to study patterns of immunoglobulin heavy chain (IgH) gene rearrangement (GR) in formalin-fixed, paraffin-embedded specimens of lymphomas and reactive conditions of mucosa-associated lymphoid tissue (MALT) and lymph node. DNA amplification was performed directly on sections obtained from paraffin blocks. Five patterns of PCR products were observed: a single band, two or more discrete bands, smearing, a single band overlying a smear, and two or more bands over a smear. A pure polyclonal pattern (smear) was observed in all of the reactive lymph nodes but in only 15% of cases of Helicobacter pylori (HP) gastritis with lymphoid hyperplasia, 25% of cases of HP gastritis without lymphoid hyperplasia, and 37% of colonic specimens of various types. Patterns consisting of multiple bands with or without background smearing were common in gastritis, colitis, and gastric lymphomas. Single bands or dominant bands were present in all lymph node and salivary gland lymphomas, 12 of 14 cases of gastric lymphoma, and 17 of 20 cases of HP gastritis with lymphoid hyperplasia. These bands were reproducible in deeper sections from the same paraffin block or similar areas sampled in different blocks in all of the lymph node and salivary gland lymphomas, 11 of 12 gastric lymphomas, but only 1 of 17 cases of HP gastritis with lymphoid hyperplasia. Bands were also found in 3 of 20 cases of HP gastritis without lymphoid hyperplasia and 17 of 38 colonic specimens, but these were not reproducible. The complexity of patterns of IgH GR in acquired MALT compared with lymph nodes may be the result of a relative paucity of B-cell clones or preferential proliferation of B-cell clones with a limited area of distribution.
A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.
In myasthenia gravis the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. AChR epitopes recognized by CD4+ T cells in myasthenic patients have been identified. AChR-specific CD4+ cell lines can be propagated by stimulation of blood lymphocytes with synthetic or biosynthetic AChR sequences. We analysed, using a semi-quantitative PCR assay, the T cell receptor (TCR) V beta usage of 16 anti-AChR polyclonal CD4+ T cell lines of known epitope specificity, propagated from myasthenic patients using pools of overlapping peptides corresponding to the sequence of an AChR subunit, or individual synthetic AChR sequences. Twelve lines had been propagated for less than 2 months, four lines for 3.5-5 months. Most lines had limited V beta usage, but in most cases different V beta regions were used for different epitopes in the same patient, and for the same epitope in different patients. In a few patients, the same V beta regions were used for recognition of different epitopes. The V beta 4 and V beta 6 regions were used most frequently. These findings suggest that the potentially autoimmune T cells that survive clonal deletion have a limited TCR repertoire. Although the present data do allow conclusions on the role of a superantigen in triggering the anti-AChR autoimmune response, the finding that different V beta regions were used in different patients does not support an important role of a superantigen in the maintenance of the CD4+ response in myasthenia gravis.
The expression of the known rat 5-HT receptor sub-types has been analyzed in a presumptive serotoninergic cell line derived from the rat raphé nuclei, using reverse transcription followed by polymerase chain reaction. By manipulating the activity of the oncogene (ts-SV40T) product used to immortalize the serotoninergic precursors, it has been possible to compare the expression of the 5-HT receptors in either replicative or differentiating cells. 5-HT1B, 5-HT1D, 5-HT3, 5-HT6 and 5-HT7 receptor gene expression were all observed in the replicating cells. However, under differentiation conditions, expression of all except the 5-HT1B receptor was lost. Only one novel amplification product appeared during early differentiation, in the 5-HT2B lane; its smaller than expected size was suggestive of a previously undescribed alternate splicing of the mRNA in brain. The curtailment of 5-HT receptor expression in differentiating neurones in vitro may reflect the normal ongoing restriction in the phenotypic potential during embryogenesis in vivo. The serotonin cells, therefore, constitute a pristine cell line in which to study the receptor pharmacology of one or more 5-HT receptor sub-types in isolation.
Single-stranded DNA aptamers that recognize DNA polymerase from Thermus acquaticus (Taq pol) with high affinity have been described recently. These aptamers have been shown to efficiently inhibit the polymerase activity of Taq pol and are useful in enhancing the amplification efficiency of low copy number targets by the polymerase chain reaction (PCR). Aptamers selected to bind to Taq pol fell into two different sequence families and inhibited several DNA polymerases isolated from the Thermus species, including that from Thermus thermophilus (Tth pol). Aptamers from one sequence family inhibited the Stoffel fragment of Taq pol efficiently, whereas those from the other family did not. Truncated aptamers derived from two parent ligands from both families were combined to form a heterodimeric aptamer that effectively inhibited all three polymerases and were shown to be useful in detecting a low copy number target by PCR amplification. These data demonstrate that the combination of aptamers with different properties into a single molecule broadens their spectrum of utility.
Healthy humans have CD4+ T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+ cells present in healthy persons. We studied CD4+ cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves' disease (GD) patients. We used short-term CD4+ cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject. We determined their TCR Vbeta usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vbeta region family, in conjunction with a constant region primer. Six lines preferentially used one Vbeta family (42-94%), different for each line. In all lines, three or less Vbeta families accounted for approximately 60% or more of the Vbeta usage. Different Vbeta regions were used by each subject. There was no obvious difference between the Vbeta usage of the lines from GD patients and healthy controls. These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4+ cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4+ cells specific for the same autoantigen in healthy subjects.
The etiology of osteoporosis is multifactorial, but there is evidence from both animal and human studies that the volume of marrow adipose tissue increases when bone volume is reduced in osteoporosis. The cell-related mechanism that may account for this inverse relationship between the volume of marrow adipose tissue and bone remains to be clarified, although it is known that both adipocytes and osteoblasts are derived from stromal cells precursors in bone marrow. We report that retroviral transduction with a temperature-sensitive oncogene (SV40 large T antigen) can generate bipotential cell lines from human marrow stroma that are capable of directed differentiation, in vitro, down either an osteogenic or adipocytic lineage pathway. One such clone, designated hOP 7, expresses type alpha 1(I) procollagen and has low alkaline phosphatase (AP) activity under basal culture conditions that is reminiscent of an osteoprogenitor cell. Exposure of hOP 7 cells to dexamethasone upregulates AP activity and enables the cells to mineralize their extracellular matrix. Also, treatment with calcitriol induces osteocalcin expression and both PTH and PGE2 induce/augment cAMP formation. Incubation with normal rabbit serum, however, causes the cells to become adipogenic as demonstrated by histological staining with Oil-red-O, expression of mRNA for the early and late adipocyte markers lipoprotein lipase and glycerol 3-phosphate dehydrogenase, respectively, and loss of type alpha 1(I) procollagen mRNA. The generation of homogeneous populations of these cells, as confirmed by Southern blot analysis, demonstrates the capacity of a human clonal cell line to differentiate in either an osteogenic or adipogenic direction.
We demonstrated previously that human skin keratinocytes express acetylcholine receptors (AChRs) sensitive to acetylcholine and nicotine, which regulate cell adhesion and motility. We demonstrate here that human and rodent bronchial epithelial cells (BECs) express AChRs similar to those expressed by keratinocytes and by some neurons. Patch-clamp experiments demonstrated that the BEC AChRs are functional, and they are activated by acetylcholine and nicotine. They are blocked by kappa-bungarotoxin, a specific antagonist of the AChR isotypes expressed by neurons in ganglia. Their ion-gating properties are consistent with those of AChR isotypes expressed in ganglia, formed by alpha3, alpha5, and beta2 or beta4 subunits. Reverse transcription-polymerase chain reaction and in situ hybridization experiments demonstrated the presence in BECs of mRNA transcripts for all those AChR subunits, both in cell cultures and in tissue sections, whereas we could not detect transcripts for the alpha2, alpha4, alpha6, and beta3 AChR subunits. The expression of alpha3 and alpha5 proteins in BEC in vivo was verified by the binding of subunit-specific antibodies to sections of trachea. Mecamylamine and kappa-bungarotoxin, which are cholinergic antagonists able to block the ganglionic alpha3 AChRs, caused a reversible change of the cell shape of cultured, confluent human BECs. This resulted in a reduction of the area covered by the cell and in cell/cell detachment. The presence of AChRs sensitive to nicotine on the lining of the airways raises the possibility that the high concentrations of nicotine resulting from tobacco smoking will cause an abnormal activation, a desensitization, or both of the bronchial AChRs. This may mediate or facilitate some of the toxic effects of cigarette smoking in the respiratory system.
In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. The anti-AChR response may originate in the thymus, which is abnormal in most MG patients and contains anti-AChR T and B cells. Microbial superantigens (sAg) may trigger autoimmune responses and in this study we sought clues as to whether sAg play a role in the pathogenesis of MG. We investigated the frequency of use of the different TCR Vbeta families by the thymus and blood T cells in MG patients and in control subjects, using a multi-primer PCR assay. Identical TCR-Vbeta usage was found in the thymi of MG patients and controls, except Vbeta2, which showed a small increase in MG patients' thymi. Blood T cells of MG patients used Vbeta4, Vbeta6, Vbeta15, Vbeta16 and Vbeta24 significantly more than those of the controls. Vbeta4 and Vbeta6 are the gene families most frequently used by anti-AChR CD4(+) cells in MG patients. Blood T cells from MG patients used Vbeta12, Vbeta14, Vbeta17 and Vbeta18 significantly less than controls. MG patients used Vbeta4 and Vbeta6 significantly more in the blood than in the thymus, while the opposite occurred for Vbeta7, Vbeta12 and Vbeta14. Controls used Vbeta17 more and Vbeta24 less in the blood than in the thymus. The preferential expansion of Vbeta4 and Vbeta6 in MG patients might reflect the immunodominance of certain AChR epitopes, or the action of a sAg outside the thymus. The minimal differences in the TCR-Vbeta usage in the blood and thymus of control subjects might be due to expansion of T cell clones specific for common antigens. Identical Vbeta usage in the thymi of MG patients and controls does not support an important role of the thymus as the location of anti-AChR sensitization when MG is clinically evident. The differences observed in the Vbeta usage in blood and thymi of MG patients are likely to be due to preferential Vbeta usage by the anti-AChR T cells in the blood.
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.
A novel method for the hot start of PCR using DNA helicases is developed. The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating. The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture.
The development of molecular diagnostic methods, particularly those utilizing PCR for the detection of parasitic protozoa will contribute greatly to the identification and control of these pathogens, by increasing speed of diagnosis, specificity and sensitivity, reproducibility and case of interpretation. PCR methods are not without their problems however, and there is a need for laboratory procedures to be refined before PCR-based assays are accepted as the tools of choice for the routine detection of protozoan parasites. The application of PCR detection to various parasites is discussed.
Over the past few years, the Australian forensic science community has adopted a common methodology and technology in the application of DNA profiling for investigative and forensic purposes. The ultimate objective of this initiative is the establishment of a national DNA database similar to that used in the UK. An integral part of this methodology is the use of "Profiler Plus," a nonaplex of STRs combined with amelogenin, a locus utilized for sex determination. This paper reports the results from a case where a mutation in the annealing region of the amelogenin primers appears to have resulted in the failure to amplify the amelogenin Y-homolog from a phenotypically normal male. The result was confirmed using two different primer sets that amplify different regions of the amelogenin gene. This situation suggests that the genetic determination of sex based on the amelogenin sequences from specimens of unknown origin, such as crime scene samples, should not be considered infallible.
Amelogenesis imperfecta (AI) is a heterogeneous group of inherited disorders of defective enamel formation. The major protein involved in enamel formation, amelogenin, is encoded by a gene located at Xp22.1-Xp22.3. This study investigated the molecular defect producing a combined phenotype of hypoplasia and hypomineralization in a family with the clinical features and inheritance pattern of X-linked amelogenesis imperfecta (XAI). Genomic DNA was prepared from buccal cells sampled from family members. The DNA was subjected to the polymerase chain-reaction (PCR) in the presence of a series of oligonucleotide primers designed to amplify all 7 exons of the amelogenin gene. Cloning and sequencing of the purified amplification products identified a cytosine deletion in exon VI at codon 119. The deletion resulted in a frameshift mutation, introducing a premature stop signal at codon 126, producing a truncated protein lacking the terminal 18 amino acids. Identifying mutations assists our understanding of the important functional domains within the gene, and finding another novel mutation emphasizes the need for family-specific diagnosis of amelogenesis imperfecta.
The epithelial or endothelial cells that line the human bronchi and the aorta express nicotinic acetylcholine receptors (nAChRs) of alpha3 subtypes. We report here that human bronchial epithelial cells (BEC) and aortic endothelial cells (AEC) express also the nAChR alpha7 subunit, which forms functional nAChRs. Polymerase chain reaction and in situ hybridization experiments detected alpha7 subunit mRNA in cultured human BEC and AEC and in sections of rat trachea. The binding of radiolabeled alpha-bungarotoxin revealed a few thousand binding sites per cell in cultured human BEC and human and bovine AEC. Western blot and immunohistochemistry experiments demonstrated that cultured BEC and AEC express a protein(s) recognized by anti-alpha7 antibodies. Whole-cell patch-clamp studies of cultured human BEC demonstrated the presence of fast-desensitizing currents activated by choline and nicotine that were blocked reversibly by methyllycaconitine (1 nM) and irreversibly by alpha-bungarotoxin (100 nM), consistent with the expression of functional alpha7 nAChRs. In some cells, choline activated also slowly decaying currents, confirming previous reports that BEC express functional alpha3beta4 nAChRs. Exposure of cultured BEC to nicotine (1 microM) for 3 days up-regulated functional alpha7 and alpha3 nAChRs, as indicated by the increased number of cells responding to acetylcholine and choline, with both fast-desensitizing currents, which were blocked irreversibly by alpha-bungarotoxin, and with slowly desensitizing currents, which are alpha-bungarotoxin-insensitive currents. The presence of alpha7 nAChRs in BEC and AEC suggests that some toxic effects of tobacco smoke could be mediated through these nicotine-sensitive receptors.
The propensity of Taq polymerase to add 3′-A overhangs (1,2) to polymerase chain reaction (PCR)-amplified DNA has made possible a simple method for cloning PCR products into a T-vector (Invitrogen, San Diego, CA) (3–5). Here, we present a related strategy that uses T-linkers to add sequences, such as restriction sites, to the ends of PCR products (see
Note 1). A single-base T overhang at the end of a synthetic double-stranded oligonucleotide linker allows ligation of the linker to the unpolished ends of a PCR product. This avoids the expense of adding the “extra” sequences to sequence-specific primers.
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