High-resolution Electrophoresis Research Articles

Enzymes produced by autoactivation of blood factor XII in buffer: A contribution from the Hematology at Biomaterial Interfaces Research Group

High-resolution electrophoresis of FXII-derived proteins produced by contact activation of FXII in buffer solutions (i.e. in absence of plasma proteins) with hydrophilic and silanized-glass activators spanning the observable range of water wettability (hydrophilic to hydrophobic), shows no evidence of proteolytic cleavage of FXII into αFXIIa or βFXIIa. The autoactivation mixture contains only a single-chain protein with a molecular weight of ∼80 kDa, confirming Oscar Ratnoff's previous finding of a single-chain activated form of FXII that he called ‘HFea’. Functional assays have shown that these autoactivation products exhibit procoagulant potential (protease activity inducing clotting of blood) or amidolytic potential (cleaves amino bonds in s-2302 chromogen but do not cause coagulation of plasma) or both amidolytic potential and procoagulant potential. Some of these proteins also have the remarkable potential to ‘suppress autoactivation’ (i.e. suppress creation of enzymes with procoagulant potential). It is thus hypothesized that autoactivation of FXII in the absence of plasma proteins generates not just a single type of activated conformer, as suggested by previous researchers, but rather an ensemble of conformer products with collective activity that varies with activator surface energy used in contact activation of FXII. Furthermore, reaction of αFXIIa with FXII in buffer solution does not produce additional αFXIIa by the putative autoamplification reaction FXIIa + FXII → 2FXIIa as has been proposed in past literature to account for the discrepancy between chromogenic and plasma-coagulation assays for αFXIIa in buffer solution. Instead, net procoagulant activity measured directly by plasma-coagulation assays, decreases systematically with increasing FXII solution concentration. Under the same reaction conditions, chromogenic assay reveals that net amidolytic activity increases with increasing FXII solution concentration. Thus, although autoamplification does not occur it appears that there is some form of “FXII self reaction” that influences products of αFXIIa reaction with FXII. Electrophoretic measurements indicate that no proteolytic cleavage takes in this reaction leading us to conclude that change in activity is most likely due to change(s) in FXII conformation (with related change in enzyme activity).

Unique self-assembly properties of a bridge-shaped protein dimer with quantum dots

How protein–protein interaction affects protein–nanoparticle self-assembly is the key to the understanding of biomolecular coating of nanoparticle in biological fluids. However, the relationship between protein shape and its interaction with nanoparticles is still under-exploited because of lack of a well-conceived binding system and a method to detect the subtle change in the protein–nanoparticle assemblies. Noticing this unresolved need, we cloned and expressed a His-tagged SpeA protein that adopts a bridge-shaped dimer structure, and utilized a high-resolution capillary electrophoresis method to monitor assembly formation between the protein and quantum dots (QDs, 5 nm in diameter). We observed that the bridge-shaped structure rendered a low SpeA:QD stoichiometry at saturation. Also, close monitoring of imidazole (Im) displacement of surface-bound protein revealed a unique two-step process. High-concentration Im could displace surface-bound SpeA protein and form a transient QD–protein intermediate, through a kinetically controlled displacement process. An affinity-driven equilibrium step then followed, resulting in re-assembling of the QD–protein complex in about 1 h. Through a temporarily formed intermediate, Im causes a rearrangement of His-tagged proteins on the surface. Thus, our work showcases that the synergistic interplay between QD–His-tag interaction and protein–protein interaction can result in unique properties of protein–nanoparticle assembly for the first time.

Urinary Screening of Mucopolysaccharidoses (MPS) in Cases Suspected of Inborn Errors of Metabolism Children in HUSM

Mucopolysaccharidoses (MPS) is a disease of inborn errors of metabolism (IEM). It constitutes a large and hetero- genous subgroup among the lysosomal storage diseases. For the detection of this disease, urinary glycosaminoglycan (GAG) is measured by dimethlmethylene blue (DMB) assay and high resolution electrophoresis (HRE) is done to characterize the different types of MPS. To screen for MPS in all urine samples suspected of inborn errors of metabolism sent to the metabolic laboratory from 1998 to 2008 and to look into the association between the positivity of DMB assay and HRE in the same sample, All urine samples sent to the metabolic laboratory that fulfilled the inclusion criteria were analyzed for GAG and were further subjected to HRE. Measurement of observed agreement using Cohen's kappa coefficient (k) was used for the association between both DMB and HRE method. A total of 134 urine samples were obtained however only 90 samples were analyzed. 28 (31.1%) of the samples had normal GAG levels, 59 (65.6%) had GAG levels between 1-2 folds above normal limits and 3 (3.3%) samples had more than 2 folds increment above the normal limits for age. Three samples showed abnormal bands when subjected to HRE. Poor association between both methods (k = 0.027) was observed. This study shows that all samples with high index of suspicion of MPS and elevated values of GAG of two folds or more should be subjected to HRE. Characterization of MPS through HRE gives the clinician some presumptive diagnosis and prognosis in managing these patients.

Abstract 5: Angiotensin II-induced Alterations of T Cell Receptor Vβ Chain Usage in Kidney and Mesenteric Vasculature

We have previously shown that T cells play a critical role in the genesis of hypertension, and that activated T cells infiltrate the vasculature and the kidney to promote vasoconstriction and sodium retention. Stimulation of the T cell receptor (TCR) with a specific antigen leads to clonal expansion of cells with identical TCRs. In contrast, if multiple antigens are presented, T cells with multiple TCRs proliferate. We sought to determine if hypertension promotes a mono- or polyclonal T cell response. TCR Vβ chain gene transcript length can be used to examine T cell clonality. We produced hypertension in C57Bl/6 mice by chronic (2 week, 490 ng/kg/min) infusion of angiotensin II and used vehicle infused mice as controls. PCR was employed to amplify the 24 TCR Vβ chain subfamilies in the kidney and mesenteric vasculature. PCR products were subjected to high-resolution capillary electrophoresis fragment analysis to determine TCR Vβ chain transcript length. Eleven of the 24 Vβ length profiles exhibited greater than 20% skewing with angiotensin II infusion in kidney and 6 of 24 exhibited greater than 20% skewing in mesentery. Dominant transcript lengths were present with angiotensin II infusion for Vβ 1 (30 ± 5 vs. 51 ± 7), 3 (29 ± 3 vs. 51 ± 12) and 20 (0 ± 0 vs. 41 ± 18) in kidney and 1 (30 ± 2 vs. 43 ± 4), 4 (27 ± 4 vs. 45 ± 3) and 20 (15 ± 3 vs. 46 ± 6) in mesentery (data are expressed as % ± S.E.M. of TCR transcripts of a given length within each Vβ family, sham vs. angiotensin II, n = 5 per group). These results suggest that hypertension results in oligoclonal T cell expansion in the kidney and mesentery. Alterations in Vβ 1 and 20 in both kidney and mesentery suggest a common TCR response between tissues.

Relationship of short tandem repeats flanking leptin-melanocortin pathway genes with anthropometric profile and leptinemia in Brazilian individuals

To investigate the relationship of short tandem repeats (STR) near genes involved in the leptin-melanocortin pathway with body mass index (BMI) and leptinemia. Anthropometric variables and leptinemia were measured in 100 obese and 110 nonobese individuals. D1S200, D2S1788, DS11912, and D18S858 loci were analyzed by PCR and high-resolution electrophoresis. Overall STR allele frequencies were similar between the obese and non-obese group (p > 0.05). Individual alleles D1S200 (17), D11S912 (43), D18S858 (11/12) were associated with obesity (p < 0.05). Individuals carrying these alleles showed higher BMI than non-carriers (p < 0.05). Moreover, a relationship between D18S858 11/12 alleles and increased waist circumference was found (p = 0.040). On the other hand, leptinemia was not influenced by the studied STRs (p > 0.05). D1S200, D11S912, and D18S858 loci are associated with increased BMI and risk for obesity in this sample.

High-resolution gel electrophoresis and sodium dodecyl sulphate–agarose gel electrophoresis on urine samples for qualitative analysis of proteinuria in dogs

The aims of the current study were to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) and high-resolution electrophoresis (HRE) can identify dogs with a urinary protein-to-creatinine ratio (UPC ratio) >0.2 and whether HRE can provide preliminary information about the type of proteinuria, using SDS-AGE as a reference method. HRE and SDS-AGE were conducted on 87 urine samples classified according to the International Renal Interest Society as non-proteinuric (NP; UPC ratio: <0.20; 32/87), borderline proteinuric (BP; UPC ratio: 0.21-0.50; 15/87), or proteinuric (P; UPC ratio: >0.51; 40/87). SDS-AGE and HRE were positive in 14 out of 32 and 3 out of 32 NP samples and in 52 out of 55 and 40 out of 55 samples with a UPC ratio >0.20, respectively. The concordance between HRE or SDS and UPC ratio was comparable (κ = 0.59; κ = 0.55). However, specificity (90%) and positive likelihood ratio (7.76) were higher for HRE than for SDS-AGE (56% and 2.16) while sensitivity was lower (73% vs. 94%). The analysis of HRE results revealed that a percentage of albumin >41.4% and an albumin/α(1)-globulin ratio (alb/α(1) ratio) >1.46 can identify samples classified by SDS-AGE as affected by glomerular proteinuria while a percentage of α(1)-globulin >40.8% and an alb/α(1) ratio <0.84 can identify samples classified by SDS-AGE as affected by tubular proteinuria. In conclusion, both SDS-AGE and HRE could misclassify samples with a UPC ratio higher or lower than 0.20. Therefore, UPC ratio must always be determined before conducting these tests. The percentage of albumin and α(1)-globulin or the alb/α(1) ratio determined by HRE can provide preliminary information about the origin of proteinuria.

Hsp90 gene, an additional target for discrimination between the potato cyst nematodes, Globodera rostochiensis and G. pallida, and the related species, G. tabacum tabacum

The heat-shock gene, Hsp90, was targeted as a new variable genomic region to supplement other DNA-based tests for identification and discrimination of Globodera pallida, G. rostochiensis and G. tabacum tabacum. Populations of the potato cyst nematodes, G. pallida and G. rostochiensis (PCN), originating from Canada, France, Belgium and USA, together with two populations of G. tabacum tabacum from the USA and France were used for the amplification of a fragment of the Hsp90 gene. General and specific primers and probes for each species were derived from the consensus and non-consensus regions of the aligned sequences, respectively. A triplex conventional PCR assay, using a general forward and reverse or three specific reverse primers, as well as a real-time PCR using general primers and specific TaqMan probes, were developed. Melting curve analysis and restriction fragment polymorphisms using high resolution electrophoresis were explored for identifying PCR amplicons that characterized and discriminated the three Globodera species in both pure and mixed samples. Results from the different molecular assay strategies confirmed the usefulness of Hsp90 as a new additional gene target and showed that several different test options could be used for discrimination of PCN.

Brody Disease: Insights Into Biochemical Features of SERCA1 and Identification of a Novel Mutation

Brody disease is an inherited disorder of skeletal muscle function characterized by increasing impairment of relaxation during exercise. The autosomal recessive form can be caused by mutations in the ATP2A1 gene, which encodes for the sarcoplasmic/endoplasmic reticulum Ca-ATPase 1 (SERCA1) protein. We studied 2 siblings affected by Brody disease. The patients complained of exercise-induced delay of muscle relaxation and stiffness since childhood and had gene analysis of ATP2A1. Morphologic and biochemical studies were performed on a muscle biopsy from 1 patient. The biopsy showed fiber size variation and increased numbers of fibers with internal nuclei. Ultrastructural examination revealed dilatation of lateral cisternae and proliferation of tubular elements of the sarcoplasmic reticulum. By immunohistochemistry, SERCA1 was expressed in a normal pattern, but sarcoplasmic reticulum Ca-ATPase activity was significantly reduced. Immunoblotting after high-resolution 2-dimensional gel electrophoresis showed a significant difference in the amount of SERCA1 protein between the patient and controls. Both patients were found to have 2 previously unreported in-frame deletions in ATP2A1. Because SERCA1 protein has specific biochemical characteristics in our patient, these results underline the importance of a pathologic and biochemical analyses for the diagnosis. In addition, we describe 2 novel mutations in the ATP2A1 gene.

TDNA-PCR Followed by Automated Fluorescent Capillary Electrophoresis for Identification of Bacterial Species

Transfer RNA intergenic spacer length polymorphism analysis (tDNA-PCR) is a simple and reproducible polymerase chain reaction (PCR) technique for identification of bacteria at the species or even subspecies level. The primers used in the PCR are based on conserved sequences located at the edges of the tRNA genes. Because the selected consensus primers are directed outwardly, the intergenic spacers are amplified rather than the genes themselves. With each PCR, several amplicons of different lengths are obtained, because several intergenic spacers are present in each bacterial genome. The patterns thus obtained are identical within species, but differ between distinct species, and as a result, can be used for identification of bacterial species. The amplicons are separated using high-resolution (1 bp) electrophoresis (e.g., fluorescent capillary electrophoresis) and immediately digitized as tables composed of numerical lengths (expressed in base pairs) and peak intensities. For identification, the resulting peak pattern can be compared with a large database of patterns of well-identified bacterial strains, using an in-house-developed software package that is available online. New patterns (linked to the correct species name, which can be obtained, e.g., after 16S rRNA gene sequence determination) can be added to expand the database further. This protocol describes tDNA-PCR, followed by automated fluorescent capillary electrophoresis to identify bacterial species.

Enhancement of DNA cleavage activity of an unnatural ferrocene-amino acid conjugate

The nucleolytic activity of an unnatural ferrocenyl amino acid can be significantly enhanced under reducing or Fenton conditions while exploiting different mechanisms of DNA cleavage. In addition, results from high resolution electrophoresis indicate that the ferrocenyl amino acid exhibits non-sequence specific cleavage of the DNA backbone.

Sequence-specific alkylation of DNA by pyrrole-imiclazole polyamides through cooperative interaction

We designed and synthesized an alkylating N-methylpyrrole (Py) -N-methylimidazole (Im) polyamide and l-(chloromethyl)-5-hydroxy-l,2-dihydro-3H benz[e]indole (seco-CBI) conjugate 1. DNA alkylating activities of conjugate 1 were evaluated by high-resolution denaturing polyacrylamide gel electrophoresis with DNA fragment containing human telomere repeat sequence. These results revealed that simultaneous treatment of the DNA fragment with conjugate 1 and Distamycin A (Dist) alkylate at the 5(')-GGTTAGGGTTA-3' sequence effectively due to cooperative interaction. Moreover, this combination has achieved 11-base-pair (bp) recognition. Thus, it is suggested that the utilization of one alkylating agent possessing long Py-Im polyamide moiety and short Py-Im polyamide partner can be an expedient way to attain the extension and precision of the recognition of DNA sequence.

A refined method for molecular typing reveals that co-occurrence of PrPSc types in Creutzfeldt–Jakob disease is not the rule

Molecular typing in Creutzfeldt–Jakob disease (CJD) relies on the detection of distinct protease-resistant prion protein (PrPSc) core fragments, which differ in molecular mass or glycoform ratio. However, the definition and correct identification of CJD cases with a co-occurrence of PrPSc types remains a challenge. With antibodies recognizing a linear epitope in the octapeptide repeat PrP region, supposed to distinguish between the two major PrPSc isoforms (ie, types 1 and 2), it was recently shown that all type 2 cases display an associated band with a type 1 migration pattern, which led to the conclusion that multiple PrPSc types regularly coexist in CJD. We studied brain samples from 53 sporadic CJD and 4 variant CJD subjects using a high-resolution electrophoresis, a wide range of proteinase K (PK) activities, the ‘type 1-selective' antibody 12B2, and several unselective antibodies. We found that the type 1-like band detected by 12B2 in all CJD subtypes associated with PrPSc type 2 is not a PK-resistant PrPSc core but rather matches the physicochemical properties of partially cleaved fragments, which result from the several PK cleavage sites included in the N-terminal portion of PrPSc. Furthermore, using gels with high resolution and a relatively high PK activity, we were able to increase the detection sensitivity of either type 1 or 2, when coexisting, to amount corresponding to 3–5% of the total PrPSc signal (ie, weak band of one type/total PrPSc). Our results show that there are many pitfalls associated with the use of ‘type 1 selective' antibodies for CJD typing studies and that co-occurrence of PrPSc types in CJD is not the rule. The present results further validate the rationale basis of current CJD classification and the qualitative nature of molecular typing in CJD.

Investigation of a protein expression profile by high-resolution bidimensional electrophoresis of Trypanosoma cruzi epimastigotes

Investigation of a protein expression profile by high-resolution bidimensional electrophoresis of Trypanosoma cruzi epimastigotes

Study of Inherited Metabolic Disorders in Singapore – 13 Years Experience

Recommended by the National Advisory Council of the Disabled, the Ministry of Health of Singapore supported a nationwide study of inherited metabolic disorders (IMDs). When the 5-year project ended, investigations were provided as a diagnostic service. This paper documents our 13-year experience. Patients with symptoms suggestive of an IMD were referred. Investigations on heparinised blood and/or urine included amino acid analysis using a Beckman 6300 Amino Acid Analyser, organic acids analysis using a Hewlett- Packard gas chromatography and mass spectrometry, mucopolysaccharides quantitative assay and high-resolution electrophoresis, sugars by thin-layer chromatography. Of the 3656 patients studied from 1992 to 2005, IMDs were found in 127 (77 males; 50 females; age range, 1 day to 56 years). Their ethnic distribution was: 55.1% Chinese, 19.7% Malays, 11.0% Indians, 11.0% other races and 3.2% unknown. IMD diagnosed comprised 41 (32.3%) organic acidurias, 34 (26.8%) amino acidaemias/acidurias, 14 (11.0%) urea cycle defects, 15 (11.8%) mucopolysaccharidoses, 6 (4.7%) carbohydrate disorders and 17 (13.4%) others. Twenty-three (18.1%) cases were diagnosed during the neonatal period and 36 (28.3%) after the age of 13. Positive detection rate was 3.5% and 48 IMDs were found. Significant proportion of cases had late-onset IMDs. Early identification of IMDs permits timely management, genetic counselling and prenatal diagnosis.

The biological impact of sequence-specific DNA alkylation by pyrrole-imidazole polyamides

We have developed a series of novel DNA alkylating polyamides possessing indole linkers. Investigations using high-resolution gel electrophoresis revealed that the indole linked Py-Im polyamide alkylated at A of a targeted nine base pair matching sequence. Evaluation in human cancer cell lines revealed that the indole linked Py-Im polyamides have strong cytotoxicities. Furthermore, we showed that alkylation of the template strand of the coding region by these polyamides causes effective gene silencing.

Molecular design of DNA alkylating pyrrole-imidazole polyamides with longer recognition sequence

The sequence-specificity, and DNA alkylating activity of the conjugate 1, which consists of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) with indole linker, were investigated in the absence or presence of partner Py-Im polyamide 2. High-resolution denaturing polyacrylamide gel electrophoresis showed that the specificity of DNA alkylation by 1 modulated in the presence of partner 2. We found that sequence-specific DNA alkylation by 1 and 2 with 10 base pair (bp) match recognition sequence through heterodimer formation. This result indicates one possibility of DNA alkylation with longer recognition sequence by different two molecules.

Familial frontotemporal dementia with neuronal intranuclear inclusions is not a polyglutamine expansion disease

BackgroundMany cases of frontotemporal dementia (FTD) are familial, often with an autosomal dominant pattern of inheritance. Some are due to a mutation in the tau- encoding gene, on chromosome 17, and show an accumulation of abnormal tau in brain tissue (FTDP-17T). Most of the remaining familial cases do not exhibit tau pathology, but display neuropathology similar to patients with dementia and motor neuron disease, characterized by the presence of ubiquitin-immunoreactive (ub-ir), dystrophic neurites and neuronal cytoplasmic inclusions in the neocortex and hippocampus (FTLD-U). Recently, we described a subset of patients with familial FTD with autopsy-proven FTLD-U pathology and with the additional finding of ub-ir neuronal intranuclear inclusions (NII). NII are a characteristic feature of several other neurodegenerative conditions for which the genetic basis is abnormal expansion of a polyglutamine-encoding trinucleotide repeat region. The genetic basis of familial FTLD-U is currently not known, however the presence of NII suggests that a subset of cases may represent a polyglutamine expansion disease.MethodsWe studied DNA and post mortem brain tissue from 5 affected members of 4 different families with NII and one affected individual with familial FTLD-U without NII. Patient DNA was screened for CAA/CAG trinucleotide expansion in a set of candidate genes identified using a genome-wide computational approach. Genes containing CAA/CAG trinucleotide repeats encoding at least five glutamines were examined (n = 63), including the nine genes currently known to be associated with human disease. CAA/CAG tract sizes were compared with published normal values (where available) and with those of healthy controls (n = 94). High-resolution agarose gel electrophoresis was used to measure allele size (number of CAA/CAG repeats). For any alleles estimated to be equal to or larger than the maximum measured in the control population, the CAA/CAG tract length was confirmed by capillary electrophoresis. In addition, immunohistochemistry using a monoclonal antibody that recognizes proteins containing expanded polyglutamines (1C2) was performed on sections of post mortem brain tissue from subjects with NII.ResultsNo significant polyglutamine-encoding repeat expansions were identified in the DNA from any of our FTLD-U patients. NII in the FTLD-U cases showed no 1C2 immunoreactivity.ConclusionWe find no evidence to suggest that autosomal dominant FTLD-U with NII is a polyglutamine expansion disease.

High Resolution Protein Electrophoresis of 100 Paired Canine Cerebrospinal Fluid and Serum

This study was performed to investigate the diagnostic relevance of cerebrospinal fluid (CSF) high resolution electrophoresis. The laboratory technique was applied to 100 paired samples of canine CSF and serum, with paired samples tested during the same analytical run, as recommended in human medicine. Ninety four of the dogs had a neurological disease and 6 healthy dogs served as a control group. A strong linear correlation between CSF total protein concentration and the albumin quota (AQ) was found in the control group and in the inflammatory (infectious or noninfectious), neoplastic, and miscellaneous groups: AQ = 0.015 CSF total protein ‐ 0.102, r= 0.990. This correlation suggests that an increased CSF total protein concentration can be an indicator of blood brain barrier dysfunction. The highest median AQ value was found in the aseptic suppurative meningitis group, but no statistical differences were found between this and the other groups. The AQ, calculated with this technique, did not provide any additional information. Moreover, although unexpected, the electrophoretic profiles were not characteristic of any particular disease. In conclusion, this study did not confirm high resolution electrophoresis of paired CSF and serum samples to be a valuable ancillary diagnostic tool for canine neurological diseases.

A comparison of plucked feathers versus blood samples as DNA sources for molecular sexing

Feathers are increasingly collected as a nondestructive source of DNA for avian genetic research. Although feather samples are not optimal in some important ways than more robust blood or tissue samples, feather sampling requires less training for field workers, results in shorter handling times for the organism, generates no hazardous wastes, and requires simpler storage procedures. Along with these largely positive attributes comes a set of challenges, particularly the relatively low copy number of DNA present in feather samples. We compared the utility and reliability of feathers to the more traditional blood samples as sources of DNA for polymerase chain reaction (PCR)-based molecular sexing of Black-capped Chickadees (Poecile atricapilla). DNA from 102 individuals was extracted separately from both single rectrices and from blood samples, and the sex of each bird was then determined using standard PCR-based methods. We found complete agreement between sex determinations based on feather versus blood DNA extractions. Slight variations in lab protocols were necessary to obtain consistent results from these two DNA sources; and we briefly discuss other sources of error that could occur in feather-based molecular sexing studies. This controlled comparison of feather versus blood samples demonstrates that plucked rectrices provide a highly reliable source of DNA for molecular sexing of wild birds. SINOPSIS Se esta incrementando la modalidad de usar plumas como una fuente no destructiva de ADN para llevar a cabo investigacion genetica en aves. Aunque las muestras tomadas de plumas, en algunas instancias no son optimas que muestras mas robustas como sangre y tejido, el uso de estas no requiere adiestramiento especial, toman menos tiempo en la manipulacion del ave, no generan desperdicios peligrosos y requieren un almacenaje sencillo. Aunque hay atributos positivos en el uso de plumas, tambien existe el problema de obtener una muestra baja de ADN. Examinamos la utilidad y confiabilidad de muestras de plumas en comparacion con tecnicas mas tradicionales como obtencion de muestras de sangre como una fuente de ADN para el sexado de Poecile atricapilla utilizando PCR. A tales efectos se extrajo ADN, de rectrices, de 102 individuos y un numero similar de muestras de sangre para determinar el sexo usando PCR. Encontramos resultados similares con ambos metodos. Sin embargo, fueron necesarios pequenas variaciones en el protocolo de laboratorio para obtener resultados consistentes, de ambas fuentes de ADN. Discutimos algunas fuentes de error que pueden ocurrir cuando se utilizan plumas para la determinacion del sexo utilizando tecnicas moleculares. Este estudio demuestra que las rectrices son una fuente confiable para obtener buenas muestras de ADN y poder determinar el sexo en aves silvestres.

Alpha-Thalassemia Subtyping and the Detection of Silent Mutations by High-Resolution Fragment Analysis and DNA Sequencing.

Background: Sets of polymerase chain reaction (PCR) primers were used that make it possible to study subtypes of the most prevalent mutations that cause alpha-thalassemia (-alpha3.7 deletions). These primers (Dev and C3, Dev and C9c) can be used in PCR to amplify regions that are characteristic of the three - alpha3.7 deletion subtypes (alpha3.7i, alpha3.7ii, alpha3.7iii). These subtypes are important because the type of alpha-chain deletion can affect the level of alpha-globin production. Methods and Results: The PCR products were screened using automated high-resolution electrophoresis on a DNA sequencer. Subtypes were then identified by automated DNA sequencing. During the screening for subtypes with high-resolution fragment analysis, new fragments were detected that were slightly different from the expected size. DNA sequencing of these fragments demonstrated the presence of three mutations that had not been described or fully characterized before. Conclusions: High-resolution fragment analysis is a convenient way to detect alpha-thalassemia subtypes, and DNA sequencing of silent mutations can lead to a better understanding of the cause of these mutations.