1) The document describes a new cloning method called Sequence and Ligation-Independent Cloning (SLIC) that uses in vitro homologous recombination to assemble multiple DNA fragments into a plasmid.
2) SLIC relies on exonuclease-generated single-stranded DNA overhangs on the vector and insert fragments to facilitate homologous recombination without requiring specific sequences.
3) Experiments show SLIC can efficiently assemble up to 5 or 10 DNA fragments simultaneously and circumvents limitations of other cloning methods by not requiring specific sequences and allowing various insert-vector combinations.
The analysis of all transcripts within a cell is of essential importance. Molecular biology provides many approaches to clone RNA transcripts into cDNA. Large cDNA collections are in the public domain to serve the research community. Today, however, new high-speed sequencing methods allow a much deeper view into transcriptomes than possible by classical cloning.
Plasmids are extrachromosomal DNA molecules found in bacteria that can replicate independently of the bacterial chromosome. They contain an origin of replication and may have genes for antibiotic resistance, virulence factors, or degradation of molecules. Plasmids can be classified based on their ability to integrate into chromosomes, copy number, ability to conjugate, and compatibility with other plasmids. Common plasmids used for cloning include pBR322, pUC, and pGEM3Z. These vectors contain antibiotic resistance genes and can be used to select for recombinant plasmids using antibiotic selection or blue/white screening of beta-galactosidase activity. pGEM3Z also allows for in vitro transcription of cloned genes.
The document describes the genome of the nematode C. elegans, noting that it has two sexes, hermaphrodites and males, with sex determined chromosomally. Classical genetic techniques can be used to study C. elegans, mapping mutant genes to chromosomes. The genome project involved sequencing over 2500 cosmids and other clones to assemble the physical map and fully sequence the genome.
This document discusses cloning vectors and plasmid-based cloning vectors. It provides information on plasmids, their structure, replication, and how they are used as cloning vectors. Some key points:
- Plasmids are small DNA molecules that can replicate independently of the bacterial chromosome and are often used as cloning vectors. They exist naturally in bacteria.
- Plasmids can have different structures depending on whether their DNA forms covalently closed circles or open circles. Supercoiled plasmids separate from open circles during electrophoresis.
- Plasmids can replicate in bacteria and confer phenotypes to their host like antibiotic resistance. They are categorized based on properties like being conjugative or copy number.
-
transfection and invitro packaging of phage genomeNOMI KhanS
There are two main methods for introducing recombinant phage DNA into bacterial cells: transfection and in vitro packaging. Transfection involves mixing purified phage DNA with competent bacterial cells and inducing DNA uptake via heat shock. In vitro packaging involves using proteins from defective phage strains to package recombinant lambda DNA into phage particles outside of cells. These phage particles can then infect bacterial cells. Recombinant phages can be identified by their ability to form clear plaques on indicator plates due to inactivation of genes like lacZ or CI upon insertion of foreign DNA.
Artificial chromosomes can be constructed in vitro from defined DNA components to carry large DNA fragments stably like natural chromosomes. There are different types of artificial chromosomes including BACs, YACs, PACs, and HACs. BACs can carry up to 300 kbp of DNA and are useful for genome sequencing. YACs allow cloning of up to 500 kbp of foreign DNA in yeast cells but clones can be unstable. PACs derive from bacteriophage P1 and carry 100-300 kbp of DNA. HACs are human-sized artificial chromosomes that can act as new chromosomes in human cells, carrying therapeutic genes.
The document summarizes key developments in the structural characterization of G protein-coupled receptors (GPCRs), beginning with the 1975 structure of bacteriorhodopsin determined by Henderson and Unwin, showing its 7 transmembrane alpha helical structure. It then discusses important milestones such as the 1983 cloning of bovine rhodopsin, the first GPCR cDNA; the 1986 cloning of the beta-2 adrenergic receptor, the first non-sensory GPCR; the 1988 cloning of the 5-HT1A receptor, the first "orphan" GPCR to be deorphanized; and the 1991 crystal structure of rhodopsin, the first GPCR structure. The document concludes with the
The document summarizes key developments in the structural characterization of G protein-coupled receptors (GPCRs), beginning with the 1975 structure of bacteriorhodopsin determined by Henderson and Unwin, showing its 7 transmembrane alpha helical structure. It then discusses important milestones such as the 1983 cloning of bovine rhodopsin, the first GPCR cDNA sequence, the 1986 cloning of the beta-2 adrenergic receptor, the first non-sensory GPCR, and the 1991 crystal structure of rhodopsin, the first GPCR structure determined. The document concludes with the 2007 crystal structure of the beta-2 adrenergic receptor bound to G protein and ligand, and the 2012 Nobel Prize
The analysis of all transcripts within a cell is of essential importance. Molecular biology provides many approaches to clone RNA transcripts into cDNA. Large cDNA collections are in the public domain to serve the research community. Today, however, new high-speed sequencing methods allow a much deeper view into transcriptomes than possible by classical cloning.
Plasmids are extrachromosomal DNA molecules found in bacteria that can replicate independently of the bacterial chromosome. They contain an origin of replication and may have genes for antibiotic resistance, virulence factors, or degradation of molecules. Plasmids can be classified based on their ability to integrate into chromosomes, copy number, ability to conjugate, and compatibility with other plasmids. Common plasmids used for cloning include pBR322, pUC, and pGEM3Z. These vectors contain antibiotic resistance genes and can be used to select for recombinant plasmids using antibiotic selection or blue/white screening of beta-galactosidase activity. pGEM3Z also allows for in vitro transcription of cloned genes.
The document describes the genome of the nematode C. elegans, noting that it has two sexes, hermaphrodites and males, with sex determined chromosomally. Classical genetic techniques can be used to study C. elegans, mapping mutant genes to chromosomes. The genome project involved sequencing over 2500 cosmids and other clones to assemble the physical map and fully sequence the genome.
This document discusses cloning vectors and plasmid-based cloning vectors. It provides information on plasmids, their structure, replication, and how they are used as cloning vectors. Some key points:
- Plasmids are small DNA molecules that can replicate independently of the bacterial chromosome and are often used as cloning vectors. They exist naturally in bacteria.
- Plasmids can have different structures depending on whether their DNA forms covalently closed circles or open circles. Supercoiled plasmids separate from open circles during electrophoresis.
- Plasmids can replicate in bacteria and confer phenotypes to their host like antibiotic resistance. They are categorized based on properties like being conjugative or copy number.
-
transfection and invitro packaging of phage genomeNOMI KhanS
There are two main methods for introducing recombinant phage DNA into bacterial cells: transfection and in vitro packaging. Transfection involves mixing purified phage DNA with competent bacterial cells and inducing DNA uptake via heat shock. In vitro packaging involves using proteins from defective phage strains to package recombinant lambda DNA into phage particles outside of cells. These phage particles can then infect bacterial cells. Recombinant phages can be identified by their ability to form clear plaques on indicator plates due to inactivation of genes like lacZ or CI upon insertion of foreign DNA.
Artificial chromosomes can be constructed in vitro from defined DNA components to carry large DNA fragments stably like natural chromosomes. There are different types of artificial chromosomes including BACs, YACs, PACs, and HACs. BACs can carry up to 300 kbp of DNA and are useful for genome sequencing. YACs allow cloning of up to 500 kbp of foreign DNA in yeast cells but clones can be unstable. PACs derive from bacteriophage P1 and carry 100-300 kbp of DNA. HACs are human-sized artificial chromosomes that can act as new chromosomes in human cells, carrying therapeutic genes.
The document summarizes key developments in the structural characterization of G protein-coupled receptors (GPCRs), beginning with the 1975 structure of bacteriorhodopsin determined by Henderson and Unwin, showing its 7 transmembrane alpha helical structure. It then discusses important milestones such as the 1983 cloning of bovine rhodopsin, the first GPCR cDNA; the 1986 cloning of the beta-2 adrenergic receptor, the first non-sensory GPCR; the 1988 cloning of the 5-HT1A receptor, the first "orphan" GPCR to be deorphanized; and the 1991 crystal structure of rhodopsin, the first GPCR structure. The document concludes with the
The document summarizes key developments in the structural characterization of G protein-coupled receptors (GPCRs), beginning with the 1975 structure of bacteriorhodopsin determined by Henderson and Unwin, showing its 7 transmembrane alpha helical structure. It then discusses important milestones such as the 1983 cloning of bovine rhodopsin, the first GPCR cDNA sequence, the 1986 cloning of the beta-2 adrenergic receptor, the first non-sensory GPCR, and the 1991 crystal structure of rhodopsin, the first GPCR structure determined. The document concludes with the 2007 crystal structure of the beta-2 adrenergic receptor bound to G protein and ligand, and the 2012 Nobel Prize
This document describes a new method called DNA assembler that allows for the rapid assembly of entire biochemical pathways in a single step using in vivo homologous recombination in yeast. The method is demonstrated by assembling a 9 kb D-xylose utilization pathway (3 genes), an 11 kb zeaxanthin biosynthesis pathway (5 genes), and a 19 kb combined D-xylose and zeaxanthin pathway (8 genes), all with high efficiencies of 70-100%. DNA assembler represents an improvement over previous methods for pathway construction as it is faster, requires only simple DNA preparation and one-step yeast transformation, and can assemble larger pathways without limitations on restriction sites.
This document discusses different types of cloning vectors. It describes plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), mammalian artificial chromosomes (MACs), PUC plasmids, phase M13 vectors, cosmids, and expression vectors. Each vector type has distinct characteristics like replication origin, selectable markers, and cloning capacity. Vectors are used to isolate, identify, and express DNA fragments for various applications such as genome sequencing and protein expression.
An overview of the history of structural studies of G protein-coupled receptors, written by Prof. Tony Harmar prior to his retirement from the University of Edinburgh and as chair of the Guide to PHARMACOLOGY.
The document describes the Gateway cloning system, a molecular biology technique for efficiently transferring DNA fragments between plasmids. It involves site-specific recombination mediated by bacteriophage lambda enzymes and proprietary enzyme mixes. DNA fragments flanked by att sites can be shuttled between donor, entry, and destination vectors for functional analysis and protein expression. The process is based on two recombination reactions - BP reaction inserts DNA into an entry vector, while LR reaction transfers it to an expression vector. This technique provides a rapid and accurate method for cloning genes into multiple vectors.
The document discusses several topics related to genomics and phenotypes:
1. Genome-wide genotyping and sequencing can detect common and rare genetic variants that contribute to differences between individuals.
2. Knowledge of genetic factors like variants in VKORC1 and CYP2C9 genes can help determine the appropriate drug dose of warfarin for anticoagulation therapy in patients.
3. Targeted cancer therapies like Herceptin and various tyrosine kinase inhibitors are effective for cancers with specific "driver mutations" like EGFR mutations, which can be identified through genomic analysis of each patient's tumor.
Lambda bacteriophage can be used as a vector for cloning large DNA fragments. It has the ability to package DNA into its phage head. Lambda naturally undergoes lytic and lysogenic cycles. In the lytic cycle, genes are expressed to replicate the DNA and package it into new phage particles. In the lysogenic cycle, the DNA integrates into the host chromosome. Lambda vectors allow replacement or insertion of foreign DNA. Insertion vectors contain a single cloning site, while replacement vectors remove a "stuffer fragment" and replace it. This allows cloning of larger DNA fragments than plasmid or insertion vectors.
Neutrophil immune function and gene expression are compromised in periparturient dairy cows. Specifically, neutrophils from postpartum cows displayed decreased reactive oxygen species production, chemotaxis, phagocytosis, and extracellular trap formation compared to late pregnancy. Expression of pro-inflammatory cytokines and genes involved in reactive oxygen species production were also downregulated postpartum based on RT-PCR and RNA sequencing. Transcriptome analysis revealed clusters of downregulated genes involved in chemotaxis, NADPH oxidation, and sterol/lipid biosynthesis pathways in postpartum neutrophils. This impaired neutrophil capacity may contribute to altered immune function in periparturient dairy cows.
Cosmid Vectors, YAC and BAC Expression VectorsCharthaGaglani
1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
3. Bacterial artificial chromosomes (BACs) are DNA constructs based on fertility plasmids that can clone up to 300 kb fragments and address issues with YAC stability and recombination.
This document discusses restriction enzymes and DNA restriction. Some key points:
- Restriction enzymes cut DNA at specific recognition sequences known as restriction sites. They are used in gene cloning and other applications.
- There are three main types of restriction enzymes - Type I, II, and III - which differ in their structure, cofactors required, recognition sequences, and cleavage sites.
- Restriction enzymes play a role in bacterial defense against bacteriophages by cleaving invading phage DNA. The host bacterial DNA is protected by methylation.
- DNA cloning uses restriction enzymes to cut out a gene segment and insert it into a vector for replication in a host cell. This allows production of multiple copies of
Replication Introduction , DNA replicating Models , Meselson and Stahl Experiments , Circuler Model of DNA replication , Replication in Prokaryotes , Replication In Eukaryotes , Comparison Between Prokaryotes and Eukaryotes Replicaton and PCR (Polymerease Chain Reaction)
piggyBac stem cell engineering technology offers multiple opportunities for licensing, including research uses, vector kits, iPS cell reprogramming, iPS/ES cell engineering, cell engineering, and therapeutics. piggyBac allows for highly efficient genetic modification and reprogramming of stem cells without leaving behind mutations. It can introduce transgenes, induce pluripotent reprogramming, drive differentiation, and then excise all transgenes from the genome without footprints. This overcomes limitations of viral vectors and offers advantages for stem cell engineering applications.
The document describes a strategy for creating a genomic library using restriction enzymes. A partial digest is performed using two enzymes that cut frequently, producing random overlapping fragments around 20kb in size. These fragments are inserted into replacement vectors and packaged in vitro to create an almost completely representative library. Alternatively, a single frequent cutting enzyme like Sau3AI can be used, which creates fragments that can be readily inserted into lambda replacement vectors.
Lectut btn-202-ppt-l6. cosmids and phagemidsRishabh Jain
Cosmids are vectors that contain one or two copies of the cos sites from bacteriophage lambda. They have a high insert capacity of 30-45 kilobases and are useful for cloning large genes and conducting genetic linkage studies at the molecular level. Cosmids are packaged in vitro into phage particles without forming plaques, instead forming colonies. During packaging, background molecules without inserts or with smaller inserts are eliminated. Cosmids have a high transformation frequency.
A DNA library is a collection of DNA fragments cloned into vectors that together represent the entire genome of an organism. There are two main types of libraries: genomic DNA libraries, which contain all the organism's DNA including coding and non-coding regions, and cDNA libraries, which contain only the expressed coding regions. To create a library, DNA is extracted from cells, cut into fragments, and inserted into vectors which are then introduced into host cells. This allows amplification and isolation of specific clones for analysis.
This document describes DNA libraries and the process of creating them. It discusses genomic libraries, which contain an organism's entire genome, and cDNA libraries, which contain copies of mRNA. The key steps to create a library are: 1) isolating and fragmenting DNA, 2) cloning the fragments into vectors, 3) transforming the vectors into host cells, 4) multiplying and screening the clones to identify fragments of interest. cDNA libraries are useful for studying eukaryotic genes as they remove non-coding regions.
The document discusses the plasmid vector pBR322, which was constructed in 1977 and is one of the most commonly used cloning vectors. It describes the origins and components of pBR322, including two antibiotic resistance genes, the origin of replication, and restriction enzyme cleavage sites. The document also summarizes the construction of several derivatives of pBR322, including pBR327, pUC18, and pBR118/119, and notes their applications and advantages over the original pBR322 vector.
Plasmids are small, circular DNA structures that can replicate independently of the host chromosome. They are commonly found in bacteria and play important roles in processes like drug resistance. Plasmid replication involves the recognition of an origin of replication sequence by plasmid-encoded initiator proteins. This leads to unwinding of the DNA and assembly of a replisome complex. The replication then proceeds bidirectionally via a rolling circle mechanism, where the growing DNA strand displaces the parental strand. Replication terminates once the circular plasmid is completely duplicated.
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
Genetic Screening and Prenatal Diagnosis of Thalassemias and Hemoglobinopathies in Taiwan Today by Ching-Tien Peng, MD, MPH, Superintendent & Prof. of The Children’s Hospital, China Medical University & Hospitals, Prof. of Biotechnology, Asia University, Taichung, Taiwan
Prenatal Testing, deteksi kelainan bawaan sejak dalam kandunganHendrik Sutopo
Pengenalan mengenai prenatal diagnosis.
Memberikan gambaran sekilas mengenai cara-cara untuk mengetahui kelainan bawaan sejak janin dalam kandungan.
lebih ditujukan untuk kalangan medis.
Non Invasive Prenatal Testing (NIPT)
Genetic screening and prenatal diagnostics involve analyzing DNA, proteins, and metabolites to detect heritable diseases and conditions. There are several types of genetic testing methodologies including cytogenetic testing of chromosomes, biochemical testing of proteins, and molecular testing of DNA sequences. Prenatal screening can occur in the first or second trimester of pregnancy and may involve chorionic villus sampling, amniocentesis, or ultrasound to check the health of the developing baby. While genetic testing can enable early detection and prevention of diseases, it also carries risks of increased anxiety, distress over family relations, and feelings of guilt from positive or negative results.
This document describes a new method called DNA assembler that allows for the rapid assembly of entire biochemical pathways in a single step using in vivo homologous recombination in yeast. The method is demonstrated by assembling a 9 kb D-xylose utilization pathway (3 genes), an 11 kb zeaxanthin biosynthesis pathway (5 genes), and a 19 kb combined D-xylose and zeaxanthin pathway (8 genes), all with high efficiencies of 70-100%. DNA assembler represents an improvement over previous methods for pathway construction as it is faster, requires only simple DNA preparation and one-step yeast transformation, and can assemble larger pathways without limitations on restriction sites.
This document discusses different types of cloning vectors. It describes plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), mammalian artificial chromosomes (MACs), PUC plasmids, phase M13 vectors, cosmids, and expression vectors. Each vector type has distinct characteristics like replication origin, selectable markers, and cloning capacity. Vectors are used to isolate, identify, and express DNA fragments for various applications such as genome sequencing and protein expression.
An overview of the history of structural studies of G protein-coupled receptors, written by Prof. Tony Harmar prior to his retirement from the University of Edinburgh and as chair of the Guide to PHARMACOLOGY.
The document describes the Gateway cloning system, a molecular biology technique for efficiently transferring DNA fragments between plasmids. It involves site-specific recombination mediated by bacteriophage lambda enzymes and proprietary enzyme mixes. DNA fragments flanked by att sites can be shuttled between donor, entry, and destination vectors for functional analysis and protein expression. The process is based on two recombination reactions - BP reaction inserts DNA into an entry vector, while LR reaction transfers it to an expression vector. This technique provides a rapid and accurate method for cloning genes into multiple vectors.
The document discusses several topics related to genomics and phenotypes:
1. Genome-wide genotyping and sequencing can detect common and rare genetic variants that contribute to differences between individuals.
2. Knowledge of genetic factors like variants in VKORC1 and CYP2C9 genes can help determine the appropriate drug dose of warfarin for anticoagulation therapy in patients.
3. Targeted cancer therapies like Herceptin and various tyrosine kinase inhibitors are effective for cancers with specific "driver mutations" like EGFR mutations, which can be identified through genomic analysis of each patient's tumor.
Lambda bacteriophage can be used as a vector for cloning large DNA fragments. It has the ability to package DNA into its phage head. Lambda naturally undergoes lytic and lysogenic cycles. In the lytic cycle, genes are expressed to replicate the DNA and package it into new phage particles. In the lysogenic cycle, the DNA integrates into the host chromosome. Lambda vectors allow replacement or insertion of foreign DNA. Insertion vectors contain a single cloning site, while replacement vectors remove a "stuffer fragment" and replace it. This allows cloning of larger DNA fragments than plasmid or insertion vectors.
Neutrophil immune function and gene expression are compromised in periparturient dairy cows. Specifically, neutrophils from postpartum cows displayed decreased reactive oxygen species production, chemotaxis, phagocytosis, and extracellular trap formation compared to late pregnancy. Expression of pro-inflammatory cytokines and genes involved in reactive oxygen species production were also downregulated postpartum based on RT-PCR and RNA sequencing. Transcriptome analysis revealed clusters of downregulated genes involved in chemotaxis, NADPH oxidation, and sterol/lipid biosynthesis pathways in postpartum neutrophils. This impaired neutrophil capacity may contribute to altered immune function in periparturient dairy cows.
Cosmid Vectors, YAC and BAC Expression VectorsCharthaGaglani
1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
3. Bacterial artificial chromosomes (BACs) are DNA constructs based on fertility plasmids that can clone up to 300 kb fragments and address issues with YAC stability and recombination.
This document discusses restriction enzymes and DNA restriction. Some key points:
- Restriction enzymes cut DNA at specific recognition sequences known as restriction sites. They are used in gene cloning and other applications.
- There are three main types of restriction enzymes - Type I, II, and III - which differ in their structure, cofactors required, recognition sequences, and cleavage sites.
- Restriction enzymes play a role in bacterial defense against bacteriophages by cleaving invading phage DNA. The host bacterial DNA is protected by methylation.
- DNA cloning uses restriction enzymes to cut out a gene segment and insert it into a vector for replication in a host cell. This allows production of multiple copies of
Replication Introduction , DNA replicating Models , Meselson and Stahl Experiments , Circuler Model of DNA replication , Replication in Prokaryotes , Replication In Eukaryotes , Comparison Between Prokaryotes and Eukaryotes Replicaton and PCR (Polymerease Chain Reaction)
piggyBac stem cell engineering technology offers multiple opportunities for licensing, including research uses, vector kits, iPS cell reprogramming, iPS/ES cell engineering, cell engineering, and therapeutics. piggyBac allows for highly efficient genetic modification and reprogramming of stem cells without leaving behind mutations. It can introduce transgenes, induce pluripotent reprogramming, drive differentiation, and then excise all transgenes from the genome without footprints. This overcomes limitations of viral vectors and offers advantages for stem cell engineering applications.
The document describes a strategy for creating a genomic library using restriction enzymes. A partial digest is performed using two enzymes that cut frequently, producing random overlapping fragments around 20kb in size. These fragments are inserted into replacement vectors and packaged in vitro to create an almost completely representative library. Alternatively, a single frequent cutting enzyme like Sau3AI can be used, which creates fragments that can be readily inserted into lambda replacement vectors.
Lectut btn-202-ppt-l6. cosmids and phagemidsRishabh Jain
Cosmids are vectors that contain one or two copies of the cos sites from bacteriophage lambda. They have a high insert capacity of 30-45 kilobases and are useful for cloning large genes and conducting genetic linkage studies at the molecular level. Cosmids are packaged in vitro into phage particles without forming plaques, instead forming colonies. During packaging, background molecules without inserts or with smaller inserts are eliminated. Cosmids have a high transformation frequency.
A DNA library is a collection of DNA fragments cloned into vectors that together represent the entire genome of an organism. There are two main types of libraries: genomic DNA libraries, which contain all the organism's DNA including coding and non-coding regions, and cDNA libraries, which contain only the expressed coding regions. To create a library, DNA is extracted from cells, cut into fragments, and inserted into vectors which are then introduced into host cells. This allows amplification and isolation of specific clones for analysis.
This document describes DNA libraries and the process of creating them. It discusses genomic libraries, which contain an organism's entire genome, and cDNA libraries, which contain copies of mRNA. The key steps to create a library are: 1) isolating and fragmenting DNA, 2) cloning the fragments into vectors, 3) transforming the vectors into host cells, 4) multiplying and screening the clones to identify fragments of interest. cDNA libraries are useful for studying eukaryotic genes as they remove non-coding regions.
The document discusses the plasmid vector pBR322, which was constructed in 1977 and is one of the most commonly used cloning vectors. It describes the origins and components of pBR322, including two antibiotic resistance genes, the origin of replication, and restriction enzyme cleavage sites. The document also summarizes the construction of several derivatives of pBR322, including pBR327, pUC18, and pBR118/119, and notes their applications and advantages over the original pBR322 vector.
Plasmids are small, circular DNA structures that can replicate independently of the host chromosome. They are commonly found in bacteria and play important roles in processes like drug resistance. Plasmid replication involves the recognition of an origin of replication sequence by plasmid-encoded initiator proteins. This leads to unwinding of the DNA and assembly of a replisome complex. The replication then proceeds bidirectionally via a rolling circle mechanism, where the growing DNA strand displaces the parental strand. Replication terminates once the circular plasmid is completely duplicated.
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
Genetic Screening and Prenatal Diagnosis of Thalassemias and Hemoglobinopathies in Taiwan Today by Ching-Tien Peng, MD, MPH, Superintendent & Prof. of The Children’s Hospital, China Medical University & Hospitals, Prof. of Biotechnology, Asia University, Taichung, Taiwan
Prenatal Testing, deteksi kelainan bawaan sejak dalam kandunganHendrik Sutopo
Pengenalan mengenai prenatal diagnosis.
Memberikan gambaran sekilas mengenai cara-cara untuk mengetahui kelainan bawaan sejak janin dalam kandungan.
lebih ditujukan untuk kalangan medis.
Non Invasive Prenatal Testing (NIPT)
Genetic screening and prenatal diagnostics involve analyzing DNA, proteins, and metabolites to detect heritable diseases and conditions. There are several types of genetic testing methodologies including cytogenetic testing of chromosomes, biochemical testing of proteins, and molecular testing of DNA sequences. Prenatal screening can occur in the first or second trimester of pregnancy and may involve chorionic villus sampling, amniocentesis, or ultrasound to check the health of the developing baby. While genetic testing can enable early detection and prevention of diseases, it also carries risks of increased anxiety, distress over family relations, and feelings of guilt from positive or negative results.
This document discusses prenatal diagnosis techniques. It notes that congenital defects can be anatomical or functional, and caused by preconception or postconception factors. Prenatal diagnosis evaluates fetuses at high risk for genetic/congenital disorders, common abnormalities, or those with ultrasound findings. Noninvasive techniques include ultrasound and MRI, while invasive techniques include amniocentesis, CVS, and cordocentesis. Diagnostic findings are confirmed through karyotyping, FISH, MLPA, CGH, and sequencing.
This document discusses several types of molecular recombination: homologous recombination, site-specific recombination, and transposition. It describes reciprocal homologous recombination through the double-strand break model and nonreciprocal homologous recombination through the Fox model. Site-specific recombination is important for viral genome insertion and has small regions of homology. Transposition involves transposable elements like transposons that are mobile genetic elements.
This document discusses genetic counseling and prenatal diagnosis. It defines genetic counseling as a process to help individuals understand genetic disorders and make informed decisions. Key points covered include:
- The goals and steps of genetic counseling
- Indications for genetic counseling such as family history of disorders
- Types of genetic counseling including prenatal and pediatric
- Methods of prenatal diagnosis including ultrasound, amniocentesis and chorionic villus sampling
- Risks and procedures for different prenatal tests
1. Prenatal screening and diagnostic techniques allow for the detection of fetal chromosomal abnormalities and genetic disorders. Techniques include first and second trimester screening, cell-free DNA screening, chorionic villus sampling, amniocentesis, and cordocentesis.
2. First trimester screening incorporates factors like maternal age, nuchal translucency, and biochemical markers. Second trimester screening analyzes maternal serum markers. Cell-free DNA screening analyzes fetal DNA in maternal blood. Invasive techniques like CVS and amniocentesis allow for karyotyping.
3. Genetic counseling is recommended to discuss family histories, risk factors, screening results
Genetic screening uses techniques like karyotyping, amniocentesis, and preimplantation genetic diagnosis to detect abnormalities or predict diseases. Karyotyping examines chromosomes for changes in number or structure. Amniocentesis analyzes amniotic fluid samples for fetal DNA, while chorionic villus sampling tests placental tissue. Preimplantation genetic diagnosis screens embryos before implantation. Genetic screening can prevent birth defects but also raises issues regarding which conditions to test for and the social impact of results.
DNA recombination mechanisms are involved in DNA repair, replication, gene expression and chromosome segregation. Recombination involves the breakage and joining of DNA strands. There are three main classes of recombination: homologous recombination between similar DNA sequences, site-specific recombination occurring at particular DNA sequences, and DNA transposition where segments of DNA move to new locations. Recombination is mediated by enzymes and involves steps like strand exchange, branch migration and resolution of Holliday junction structures to produce recombinant DNA products.
Noninvasive techniques are used for prenatal screening and diagnosis. These include fetal ultrasound imaging to visualize anatomy, measuring maternal serum markers like alpha-fetoprotein to screen for neural tube defects, and separating fetal cells from the mother's bloodstream for genetic analysis. More invasive techniques provide tissue samples through procedures like amniocentesis and chorionic villus sampling for cytogenetic or molecular testing to confirm or diagnose genetic disorders.
Genetic screening counseling Prenatal Testing M Phil 17 2-15Yahya Noori, Ph.D
This document provides an overview of genetic counseling, screening, and prenatal testing. It discusses:
- The purpose of genetic screening to identify individuals at risk for genetic disorders and provide reproductive options.
- Methods of genetic counseling including gathering a family history, physical exam, and laboratory testing to establish diagnoses.
- Prenatal testing options like nuchal translucency measurement and amniocentesis to detect chromosomal abnormalities.
- Principles of genetic screening tests and the importance of follow-up diagnostic testing for positive results.
- Factors that influence genetic risk like penetrance and expressivity of traits.
- Examples of career screening programs and challenges in genetic counseling like consanguinity.
DNA repair is a collection of processes cells use to identify and correct damage to DNA. Failure to repair damaged DNA leads to mutations, which are permanent changes in the DNA sequence. There are several types of DNA damage including mismatches, modified DNA bases, and single or double strand breaks. Cells use multiple repair pathways like direct reversal, base excision repair, nucleotide excision repair, recombination repair, and translesion DNA synthesis to fix different types of DNA damage. Homologous recombination repairs double strand breaks by exchanging DNA between similar molecules, while site-specific and transposon recombination involve movement of DNA between defined sequences.
This document discusses genetic recombination in bacteria. It defines several key terms and describes three main processes of genetic recombination in prokaryotes: transformation, transduction, and conjugation. Transformation involves donor DNA being taken up from the environment, transduction involves DNA transfer mediated by a virus, and conjugation involves cell-to-cell contact and plasmid transfer. The document also discusses Griffith's experiment, which provided early evidence of bacterial transformation, and the role of RecA protein in general recombination between homologous DNA sequences.
This document discusses genetic testing, including definitions, purposes, types, and techniques. It covers several types of genetic testing such as carrier screening, preimplantation diagnosis, prenatal testing, newborn screening, and predictive adult testing. Techniques discussed include amniocentesis, chorionic villus sampling, fetal cell analysis from maternal blood, analysis of maternal serum markers, ultrasound, autopsy, and culture/microscopy. Ethical considerations around informed consent are also mentioned.
Prenatal diagnosis provides couples at risk of genetic disorders with informed choices. Non-invasive tests like maternal serum screening and ultrasound can detect abnormalities, while invasive tests like amniocentesis and chorionic villus sampling allow for chromosome analysis but carry small risks of miscarriage. Advanced maternal age is the most common indication for testing. Prenatal diagnosis aims to detect issues prenatally in order to provide counseling, reduce anxiety, and potentially enable prenatal treatment in the future through gene therapy.
The document discusses various types of DNA damage including deamination, depurination, UV light-induced T-T and T-C dimers, alkylation, oxidative damage, replication errors, and double-strand breaks. It then summarizes different DNA repair pathways such as base excision repair, nucleotide excision repair, mismatch repair, direct repair, recombination repair, and non-homologous end-joining. The SOS response in bacteria is also summarized as activating error-prone repair when normal repair pathways are overwhelmed.
Genome editing with engineered nucleasesKrishan Kumar
Genome editing uses engineered nucleases to insert, replace or remove DNA from the genome. These nucleases create targeted double-strand breaks which are repaired through natural DNA repair processes, allowing for changes to the genome sequence. Three main engineered nuclease systems for genome editing are ZFNs, TALENs, and CRISPR-Cas9. CRISPR uses a guide RNA and Cas9 nuclease to make precise cuts at targeted DNA sequences for editing. It has advantages over ZFNs and TALENs in being cheaper, easier to design, and more efficient. Genome editing holds promise for applications in crops, medicine, and research.
This document summarizes information about the CRISPR Cas9 genome editing tool. It discusses how CRISPR Cas9 uses guide RNA and the Cas9 enzyme to create targeted double-strand breaks in DNA, allowing genes to be knocked out or altered. The document outlines the history and mechanism of CRISPR Cas9, compares it to other genome editing tools, discusses its applications in plant breeding including reducing off-target effects, and provides an example of using it to create parthenocarpic tomato plants.
CRISPR/Cas9 is an advanced genome editing technology that can be used to develop plant disease resistance. It involves a Cas9 enzyme that acts like molecular scissors to cut DNA at specific locations guided by CRISPR RNA. This triggers DNA repair that can introduce changes to genes. Researchers have used CRISPR/Cas9 to develop resistance in plants against viruses, fungi, and bacteria by editing genes involved in host-pathogen interaction and disease susceptibility. It provides a precise and efficient way to edit plant genomes to improve crop resistance compared to previous tools. Scientists continue working to enhance the specificity and control of CRISPR/Cas9 for genome editing applications in agriculture.
This document discusses various types of vectors used for cloning, including bacteriophage vectors, plasmid vectors, cosmid vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and shuttle vectors. Key points include:
- Bacteriophage derivatives like lambda phage are suitable for cloning large eukaryotic DNA due to abilities like packaging millions of clones and size selection of DNA.
- Phage-based vectors can be insertional, containing a single cloning site, or replacement vectors with two cloning sites allowing DNA substitution.
- Cosmids are hybrid phage-plasmid vectors that can package DNA up to 48 kb into phage particles.
- Y
DNA-Protein interaction by 3C based method.pptxKashvi Jadia
This ppt includes different 3C based techniques for study of DNA-Protein interaction. Data from given research papers are taken for education purpose only,.
Genetic engineering involves the direct manipulation of genes through techniques like gene cloning and transfer. Key tools include restriction enzymes, plasmids, and the creation of DNA libraries which allow genes to be identified and isolated. Techniques like PCR, gel electrophoresis, and blotting allow analysis and study of DNA, RNA, and proteins. Restriction mapping uses restriction enzymes to cut DNA into fragments which can then be used to identify genes and construct maps of DNA sequences.
The document discusses DNA cloning techniques. It describes how DNA cloning allows for the mass amplification and stable propagation of specific DNA sequences. The key steps involve using restriction enzymes to cut DNA fragments and plasmids, ligating the DNA fragment into the plasmid, transforming host cells with the recombinant plasmid, and selecting for transformed cells. Plasmids are commonly used as cloning vectors due to their small size, circular structure, and ability to replicate independently of the host cell genome.
CRISPR is a new mechanism\tool to edit genes and in coming future it will provide us many new levels of success in curing of genetic disorders and modifying genes for human benifit
Molecular cloning refers to the process of replicating DNA molecules to generate multiple identical copies. The key steps involve choosing a host organism and vector, preparing the vector and DNA to be cloned, creating recombinant DNA, introducing it into host cells, selecting cells containing the recombinant DNA, and screening clones. Applications include studying gene expression, producing recombinant proteins, creating transgenic organisms, and developing gene therapies.
Molecular cloning refers to the process of replicating DNA molecules to generate multiple identical copies. It involves isolating a gene or DNA fragment and inserting it into a host organism to generate multiple copies. The key steps include choosing a host and vector, preparing the vector and DNA to be cloned, creating recombinant DNA, introducing it into a host, selecting clones containing the insert, and screening clones. Applications include studying gene expression, producing recombinant proteins, creating transgenic organisms, and developing gene therapies.
Molecular cloning refers to the process of replicating DNA molecules to generate multiple identical copies. It involves isolating a gene or DNA fragment and inserting it into a host organism to generate multiple copies. The key steps include choosing a host and vector, preparing the vector and DNA to be cloned, creating recombinant DNA, introducing it into a host, selecting clones containing the insert, and screening clones. Applications include studying gene expression, producing recombinant proteins, creating transgenic organisms, and developing gene therapies.
Gene libraries, such as cDNA and genomic libraries, allow isolation of specific genes. cDNA libraries contain only exons and reflect gene expression levels, while genomic libraries contain all DNA fragments. Libraries are constructed by fragmenting DNA and cloning into vectors before transforming bacteria. They can be screened by hybridization, PCR, or immunological assays to detect gene products. Common steps include lysis, fixation, and detection to identify positive clones containing genes of interest.
Genetic recombination involves the breaking and rejoining of DNA to form new combinations of genes. It occurs primarily during meiosis through several types of recombination, including homologous recombination where DNA exchanges occur between similar DNA molecules. This increases genetic diversity and allows for traits to be mixed. Recombination benefits populations by generating variety among offspring and allowing deleterious genes to be removed without losing the entire chromosome. It has applications in cloning, mapping genes, and making transgenic organisms.
Gene Cloning Vectors - Plasmids, Bacteriophages and Phagemids.Ambika Prajapati
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium.
They allow the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
Types -
1.Plasmid vectors.
2.Bacteriophage vectors .
3.Phagemids.
This presentation deals with the introduction of Recombinant DNA Technology. The role of different enzymes. Specifically Restriction endonucleases and roles of various vectors.
Recombinant DNA technology allows scientists to isolate, amplify, and manipulate genes. It involves inserting a fragment of DNA into a vector, such as a plasmid, and introducing it into a host cell. This causes the gene of interest to be replicated in large quantities. Vectors contain an origin of replication, antibiotic resistance genes, and sites for inserting DNA. Common vectors include plasmids, bacteriophages, cosmids, BACs, and YACs, which can accommodate varying sizes of DNA inserts. Recombinant DNA is used to study gene structure and function, produce therapeutic proteins, and correct genetic defects.
This document provides an overview of CRISPR/Cas9 gene editing technology. It discusses the history of CRISPR discoveries from 1993 onwards and how the technology has been adapted for genome editing. Previous gene editing methods like ZFNs, TALENs and rAAVs are also summarized. The document then explains in detail how the CRISPR/Cas9 system works to edit genomes using a Cas9 enzyme guided by CRISPR RNA. Applications of CRISPR like genomic editing through knockouts and gene silencing are highlighted.
Thinking of getting a dog? Be aware that breeds like Pit Bulls, Rottweilers, and German Shepherds can be loyal and dangerous. Proper training and socialization are crucial to preventing aggressive behaviors. Ensure safety by understanding their needs and always supervising interactions. Stay safe, and enjoy your furry friends!
How to Build a Module in Odoo 17 Using the Scaffold MethodCeline George
Odoo provides an option for creating a module by using a single line command. By using this command the user can make a whole structure of a module. It is very easy for a beginner to make a module. There is no need to make each file manually. This slide will show how to create a module using the scaffold method.
A workshop hosted by the South African Journal of Science aimed at postgraduate students and early career researchers with little or no experience in writing and publishing journal articles.
This presentation was provided by Steph Pollock of The American Psychological Association’s Journals Program, and Damita Snow, of The American Society of Civil Engineers (ASCE), for the initial session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session One: 'Setting Expectations: a DEIA Primer,' was held June 6, 2024.
The simplified electron and muon model, Oscillating Spacetime: The Foundation...RitikBhardwaj56
Discover the Simplified Electron and Muon Model: A New Wave-Based Approach to Understanding Particles delves into a groundbreaking theory that presents electrons and muons as rotating soliton waves within oscillating spacetime. Geared towards students, researchers, and science buffs, this book breaks down complex ideas into simple explanations. It covers topics such as electron waves, temporal dynamics, and the implications of this model on particle physics. With clear illustrations and easy-to-follow explanations, readers will gain a new outlook on the universe's fundamental nature.
it describes the bony anatomy including the femoral head , acetabulum, labrum . also discusses the capsule , ligaments . muscle that act on the hip joint and the range of motion are outlined. factors affecting hip joint stability and weight transmission through the joint are summarized.
This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
Physiology and chemistry of skin and pigmentation, hairs, scalp, lips and nail, Cleansing cream, Lotions, Face powders, Face packs, Lipsticks, Bath products, soaps and baby product,
Preparation and standardization of the following : Tonic, Bleaches, Dentifrices and Mouth washes & Tooth Pastes, Cosmetics for Nails.