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Papers by Alexander Kuklin

Biochimica et biophysica acta (N), Oct 1, 1999

In Vitro Cellular & Developmental Biology - Plant

Biotechnology & Biotechnological Equipment, 1998

DNA array technology makes it possible to simultaneously study the expression of thousands of gen... more DNA array technology makes it possible to simultaneously study the expression of thousands of genes in a single experiment. DNA arrays are microscope slides (microarrays), or membrane filters (macroarrays) containing a large number of immobilized DNA samples. An array of cDNA-spots is subsequently probed with labeled cDNAs, which are obtained by reverse-transcriptase reaction from total RNA pools corresponding to the test and reference biological sources. Following the above hybridization step with dye-tagged or radioactively labeled probes, the DNA array is scanned to generate two images, each corresponding to one of the dye “colors.” A DNA array project typically requires iterations of series of processes, starting from experiment design and array fabrication, through array scanning, image analysis, and finally gene expression data analysis. In the maturation process of microarray technology, there are two kinds of challenge. One is to develop the hardware for conducting hybridiza...

BioTechniques, 1999

Cloning involves the ligation of a single DNA molecule into a single plasmid vector (1). A freque... more Cloning involves the ligation of a single DNA molecule into a single plasmid vector (1). A frequent target for cloning are polymerase chain reaction (PCR) products. Primer-dimers are contaminants inherent to PCR and yet suitable for cloning. A more serious problem is multiple PCR products that are of similar size and with similar migratory properties during electrophoresis. Conventionally, PCR products are analyzed on an agarose gel before use in ligation reactions to detect the presence of multiple products and primer-dimers. The ability to distinguish between multiple products with similar size and migratory properties depends on the resolution and sensitivity of the method used for PCR product analysis. Failing to detect the heterogeneous nature of a PCR product inevitably leads to a heterogeneous population of clones and the concomitant need to analyze a larger number of clones. Here, we used ion-pair, reversedphase HPLC (IP-RP-HPLC) combined with cloning and sequencing to characterize a PCR product that yielded a single band during gel electrophoresis. IP-RP-HPLC, which allows for a sizebased separation of DNA fragments us

…, Jan 1, 2003

The DNA microarray technology has arguably caught the attention of the worldwide life science com... more The DNA microarray technology has arguably caught the attention of the worldwide life science community and is now systematically supporting major discoveries in many fields of study. The majority of the initial technical challenges of conducting experiments are being resolved, only to be replaced with new informatics hurdles, including statistical analysis, data visualization, interpretation, and storage. Two systems of databases, one containing expression data and one containing annotation data are quickly becoming essential knowledge repositories of the research community. This present paper surveys several databases, which are considered "pillars" of research and important nodes in the network. This paper focuses on a generalized workflow scheme typical for microarray experiments using two examples related to cancer research. The workflow is used to reference appropriate databases and tools for each step in the process of array experimentation. Additionally, benefits and drawbacks of current array databases are addressed, and suggestions are made for their improvement.

Background: The agouti protein is a paracrine factor that is normally present in the skin of many... more Background: The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals.
Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1
receptor (Mc1r) on melanocytes. Lethal yellow (Ay) and viable yellow (Avy) are dominant regulatory mutations in the mouse
agouti gene that cause the wild-type protein to be produced at abnormally high levels throughout the body. Mice
harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia,
hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver.
The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to
recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels
of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in
the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed
by its ability to inhibit binding of the α-melanocyte stimulating hormone (αMSH) to the Mc1r. Body weight, plasma insulin
and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a
single intraperitoneal injection (10 mg/kg) of the hepatocellular carcinogen, diethylnitrosamine (DEN), at 15 days of age.
Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights
were recorded.
Results: The albumin-agouti transgene was expressed at high levels in the livers of mice and produced a functional agouti
protein. Albumin-agouti transgenic mice had normal body weights and normal levels of blood glucose and plasma insulin,
but responded to chemical initiation of the liver with an increased number of liver tumors compared to non-transgenic
control mice.
Conclusions: The data demonstrate that liver-specific expression of the agouti gene is not sufficient to induce obesity
or diabetes, but, in the absence of these factors, agouti continues to promote hepatocellular carcinogenesis.

Biotechnology & Biotechnological Equipment, 1990

Http Dx Doi Org 10 1089 Gte 1997 1 201, Mar 31, 2009

Molecular Biotechnology, 1999

A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system f... more A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system for DNA fragment analysis is described. The technique is used for screening of unknown mutations or polymorphisms. Fragments are isolated during the analysis from the system by an automated unit for high-throughput projects or manually when working with a few samples.

Molecular Biotechnology, 1999

A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system f... more A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system for DNA fragment analysis is described. The technique is used for screening of unknown mutations or polymorphisms. Fragments are isolated during the analysis from the system by an automated unit for high-throughput projects or manually when working with a few samples.

Molecular and Cellular Probes, 1999

The ability to rapidly and reliably genotype mice is an important concern. Traditional methods em... more The ability to rapidly and reliably genotype mice is an important concern. Traditional methods employ labour intensive and time consuming techniques such as test crossing, gel electrophoresis or nucleic acid hybridization. Here we show that a new molecular biology workstation, the WAVE DNA Fragment Analysis System, can easily resolve polymerase chain reaction (PCR) products that have small differences in their lengths. Analysis is fully automated and takes less than 7 min per sample. Approximately 200 samples can be analysed per day with only minutes of hands-on time after completion of the PCR. Genotyping with the WAVE DNA Fragment Analysis System is a fast and efficient method with minimal manual intervention.

American Journal of Medical Genetics, 2000

Briefings in Functional Genomics and Proteomics, 2003

Genetica, 2000

This paper focuses on microarray image analysis and discusses a completely automated approach to ... more This paper focuses on microarray image analysis and discusses a completely automated approach to image processing, which eliminates human intervention. A system for automated image processing is described, which is capable of processing image files in a batch-mode thus allowing high-throughput of microarray image analysis. Grid-placement and spot finding are achieved without operator's help. The software eliminates noise signals from the data analysis process and minimizes operator's involvement in the procedure.

Frontiers in Neuroscience, 2001

Plant Cell, Tissue and Organ Culture, 1994

Journal of the Association for Laboratory Automation, 2000

DNA array technology makes it possible to simultaneously study the expression of thousands of gen... more DNA array technology makes it possible to simultaneously study the expression of thousands of genes in a single experiment. DNA arrays are microscope slides (microarrays), or membrane filters (macroarrays) containing a large number of immobilized DNA samples. An array of ...

Biochimica et biophysica acta (N), Oct 1, 1999

In Vitro Cellular & Developmental Biology - Plant

Biotechnology & Biotechnological Equipment, 1998

DNA array technology makes it possible to simultaneously study the expression of thousands of gen... more DNA array technology makes it possible to simultaneously study the expression of thousands of genes in a single experiment. DNA arrays are microscope slides (microarrays), or membrane filters (macroarrays) containing a large number of immobilized DNA samples. An array of cDNA-spots is subsequently probed with labeled cDNAs, which are obtained by reverse-transcriptase reaction from total RNA pools corresponding to the test and reference biological sources. Following the above hybridization step with dye-tagged or radioactively labeled probes, the DNA array is scanned to generate two images, each corresponding to one of the dye “colors.” A DNA array project typically requires iterations of series of processes, starting from experiment design and array fabrication, through array scanning, image analysis, and finally gene expression data analysis. In the maturation process of microarray technology, there are two kinds of challenge. One is to develop the hardware for conducting hybridiza...

BioTechniques, 1999

Cloning involves the ligation of a single DNA molecule into a single plasmid vector (1). A freque... more Cloning involves the ligation of a single DNA molecule into a single plasmid vector (1). A frequent target for cloning are polymerase chain reaction (PCR) products. Primer-dimers are contaminants inherent to PCR and yet suitable for cloning. A more serious problem is multiple PCR products that are of similar size and with similar migratory properties during electrophoresis. Conventionally, PCR products are analyzed on an agarose gel before use in ligation reactions to detect the presence of multiple products and primer-dimers. The ability to distinguish between multiple products with similar size and migratory properties depends on the resolution and sensitivity of the method used for PCR product analysis. Failing to detect the heterogeneous nature of a PCR product inevitably leads to a heterogeneous population of clones and the concomitant need to analyze a larger number of clones. Here, we used ion-pair, reversedphase HPLC (IP-RP-HPLC) combined with cloning and sequencing to characterize a PCR product that yielded a single band during gel electrophoresis. IP-RP-HPLC, which allows for a sizebased separation of DNA fragments us

…, Jan 1, 2003

The DNA microarray technology has arguably caught the attention of the worldwide life science com... more The DNA microarray technology has arguably caught the attention of the worldwide life science community and is now systematically supporting major discoveries in many fields of study. The majority of the initial technical challenges of conducting experiments are being resolved, only to be replaced with new informatics hurdles, including statistical analysis, data visualization, interpretation, and storage. Two systems of databases, one containing expression data and one containing annotation data are quickly becoming essential knowledge repositories of the research community. This present paper surveys several databases, which are considered "pillars" of research and important nodes in the network. This paper focuses on a generalized workflow scheme typical for microarray experiments using two examples related to cancer research. The workflow is used to reference appropriate databases and tools for each step in the process of array experimentation. Additionally, benefits and drawbacks of current array databases are addressed, and suggestions are made for their improvement.

Background: The agouti protein is a paracrine factor that is normally present in the skin of many... more Background: The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals.
Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1
receptor (Mc1r) on melanocytes. Lethal yellow (Ay) and viable yellow (Avy) are dominant regulatory mutations in the mouse
agouti gene that cause the wild-type protein to be produced at abnormally high levels throughout the body. Mice
harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia,
hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver.
The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to
recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels
of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in
the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed
by its ability to inhibit binding of the α-melanocyte stimulating hormone (αMSH) to the Mc1r. Body weight, plasma insulin
and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a
single intraperitoneal injection (10 mg/kg) of the hepatocellular carcinogen, diethylnitrosamine (DEN), at 15 days of age.
Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights
were recorded.
Results: The albumin-agouti transgene was expressed at high levels in the livers of mice and produced a functional agouti
protein. Albumin-agouti transgenic mice had normal body weights and normal levels of blood glucose and plasma insulin,
but responded to chemical initiation of the liver with an increased number of liver tumors compared to non-transgenic
control mice.
Conclusions: The data demonstrate that liver-specific expression of the agouti gene is not sufficient to induce obesity
or diabetes, but, in the absence of these factors, agouti continues to promote hepatocellular carcinogenesis.

Biotechnology & Biotechnological Equipment, 1990

Http Dx Doi Org 10 1089 Gte 1997 1 201, Mar 31, 2009

Molecular Biotechnology, 1999

A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system f... more A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system for DNA fragment analysis is described. The technique is used for screening of unknown mutations or polymorphisms. Fragments are isolated during the analysis from the system by an automated unit for high-throughput projects or manually when working with a few samples.

Molecular Biotechnology, 1999

A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system f... more A procedure for isolation of polymerase chain reaction (PCR) fragments from an automated system for DNA fragment analysis is described. The technique is used for screening of unknown mutations or polymorphisms. Fragments are isolated during the analysis from the system by an automated unit for high-throughput projects or manually when working with a few samples.

Molecular and Cellular Probes, 1999

The ability to rapidly and reliably genotype mice is an important concern. Traditional methods em... more The ability to rapidly and reliably genotype mice is an important concern. Traditional methods employ labour intensive and time consuming techniques such as test crossing, gel electrophoresis or nucleic acid hybridization. Here we show that a new molecular biology workstation, the WAVE DNA Fragment Analysis System, can easily resolve polymerase chain reaction (PCR) products that have small differences in their lengths. Analysis is fully automated and takes less than 7 min per sample. Approximately 200 samples can be analysed per day with only minutes of hands-on time after completion of the PCR. Genotyping with the WAVE DNA Fragment Analysis System is a fast and efficient method with minimal manual intervention.

American Journal of Medical Genetics, 2000

Briefings in Functional Genomics and Proteomics, 2003

Genetica, 2000

This paper focuses on microarray image analysis and discusses a completely automated approach to ... more This paper focuses on microarray image analysis and discusses a completely automated approach to image processing, which eliminates human intervention. A system for automated image processing is described, which is capable of processing image files in a batch-mode thus allowing high-throughput of microarray image analysis. Grid-placement and spot finding are achieved without operator's help. The software eliminates noise signals from the data analysis process and minimizes operator's involvement in the procedure.

Frontiers in Neuroscience, 2001

Plant Cell, Tissue and Organ Culture, 1994

Journal of the Association for Laboratory Automation, 2000

DNA array technology makes it possible to simultaneously study the expression of thousands of gen... more DNA array technology makes it possible to simultaneously study the expression of thousands of genes in a single experiment. DNA arrays are microscope slides (microarrays), or membrane filters (macroarrays) containing a large number of immobilized DNA samples. An array of ...