Introduction to Gene Cloning and Analysis

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en-us LUSNKO4EH Gene Cloning and Analysis Introduction to Gene Cloning and Analysis /webroot/web/html/lsr/solutions/applications/genomics <p>Gene cloning is a common practice in molecular biology labs that is used by researchers to create copies of a particular gene for downstream applications, such as sequencing, mutagenesis, genotyping or heterologous expression of a protein. The traditional technique for gene cloning involves the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element, such as a bacterial plasmid. This technique is commonly used today for isolating long or unstudied genes and protein expression. A more recent technique is the use of <a href="/evportal/destination/solutions?catID=LUSNYI15">polymerase chain reaction (PCR)</a> for amplifying a gene of interest. The advantage of using PCR over traditional gene cloning, as described above, is the decreased time needed for generating a pure sample of the gene of interest. However, gene isolation by PCR can only amplify genes with predetermined sequences. For this reason, many unstudied genes require initial gene cloning and sequencing before PCR can be performed for further analysis.</p> <p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSNINKSY">Gene Expression Analysis,</a> <a href="/evportal/destination/solutions?catID=LUSNLMLPT">Mutational Analysis,</a> and <a href="/evportal/destination/solutions?catID=LUSNMFHYP">Epigenetics and Chromatin Structure.</a></p> DNA Sequencing <p><a name="dna_sequencing"></a>DNA sequencing is typically the first step in understanding the genetic makeup of an organism, which helps to:</p> <ul> <li>Locate regulatory and gene sequences</li> <li>Compare homologous genes across species</li> <li>Identify mutations</li> </ul> <p>Sequencing uses biochemical methods to determine the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a DNA oligonucleotide. Knowing the sequence of a particular gene will assist in further analysis to understand the function of the gene. <a href="/evportal/destination/solutions?catID=LUSOBDHYP">PCR</a> is used to amplify the gene of interest before sequencing can be performed. Many biotechnology companies offer sequencing instruments, however, these instruments can be expensive. As a result, many researchers usually perform PCR in-house and then send out their samples to sequencing labs.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Site-Directed Mutagenesis <p><a name="site_directed"></a>Site-directed mutagenesis is a widely used procedure for the study of the structure and function of proteins by modifying the encoding DNA. By using this method, mutations can be created at any specific site in a gene whose wild-type sequence is already known. Many techniques are available for performing site-directed mutagenesis. A classic method for introducing mutations, either single base pairs or larger insertions, deletions, or substitutions into a DNA sequence, is the Kunkel method.</p> <p>The first step in any site-directed mutagenesis method is to clone the gene of interest. For the Kunkel method, the cloned plasmid is then transformed into a dut ung mutant of <em>Escherichia coli</em>. This <em>E. coli</em> strain lacks dUTPase and uracil deglycosidase, which will ensure that the plasmid containing the gene of interest will be converted to DNA that lacks Ts and contains Us instead.</p> <p>The next step is to design a primer that contains the region of the gene which you wish to mutate, along with the mutation you want to introduce. PCR can then be used with the mutated primers to create hybrid plasmids; each plasmid will now contain one strand without the mutation and uracil bases, and another strand with the mutation and lacking uracil.</p> <p>The final step is to isolate this hybrid plasmid and transform it into a different strain that does contain the uracil-DNA glycosylase (ung) gene. The uracil deglycosidase will destroy the strands that contain uracil, leaving only the strands with your mutation. When the bacteria replicate, the resulting plasmids will contain the mutation on both strands.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Genotyping <p>Genotyping is the process of determining the DNA sequence specific to an individual's genotype. This process can be accomplished by several techniques, such as high resolution melt (<a href="/evportal/destination/solutions?catID=LUSOIH97Q">HRM</a>) analysis, or any other <a href="/evportal/destination/solutions?catID=LUSNLMLPT">mutation detection technique</a>. All of these techniques will provide an insight into the individual's genotype, which can help determine specific sequences that can be manipulated and cloned for further analysis.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Heterologous Protein Expression <p>Heterologous protein expression uses gene cloning to express a protein of interest in a self-replicating genetic element, such as a bacterial plasmid. Heterologous expression is used to produce large amounts of a protein of interest for functional and biochemical analyses.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Further Reading <p>Araya-Garay JM et al. (2011). cDNA cloning of a novel gene codifying for the enzyme lycopene &beta;-cyclase from <em>Ficus carica</em> and its expression in <em>Escherichia coli</em>. Appl Microbiol Biotechnol 92, 769&ndash;777. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/21792589" target="_blank">21792589</a></p> <p>Eckert C et al. (2006). DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother 57, 14&ndash;23. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16291869" target="_blank">16291869</a></p> <p>Handa P and Varshney U (1998). Rapid and reliable site-directed mutagenesis using Kunkel's approach. Indian J Biochem Biophys 35, 63&ndash;66. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/9753863" target="_blank">9753863</a></p> <p>Hanner M et al. (1996). Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc Natl Acad Sci USA 93, 8072&ndash;8077. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/8755605" target="_blank">8755605</a></p> <p>Kerovuo J et al. (1998). Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from <em>Bacillus subtilis</em>. Appl Environ Microbiol 64, 2079&ndash;2085. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/9603817" target="_blank">9603817</a><br /> <br /> Lauretti L et al. (1999). Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a <em>Pseudomonas aeruginosa</em> clinical isolate. J Antimicrob Chemother 43, 1584&ndash;1590. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/10390207" target="_blank">10390207</a></p> <p>Morales G et al (2010). Resistance to linezolid is mediated by the cfr gene in the first report of an outbreak of linezolid-resistant <em>Staphylococcus aureus</em>. Clin Infect Dis 50, 821&ndash;825. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/20144045" target="_blank">20144045</a></p> <p>Naldini L et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263&ndash;267. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/8602510" target="_blank">8602510</a></p> <p>Riordan JR et al. (1999). Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066&ndash;1073. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2475911" target="_blank">2475911</a></p> <div class="top"><a href="#helptop">Back to Top</a></div> 2249 Examples of Electro-Transfected Eukaryotic and Prokaryotic Cells, Rev A 2249 /webroot/web/pdf/lsr/literature/Bulletin_2249.pdf Literature PDF Other /webroot/web/images/general/icons/icon_pdf.gif No Examples of Electro-Transfected Eukaryotic and Prokaryotic Cells, Rev A 2249 lit2249, bulletin 2249, transfection, trans-fection, transformation, genepulser, gene pulser, electroporation, electroporator, 165-2660, 165-2662, condition, protocol, shockpod, 1652661, efficiency, protocol, protocols, 1652081, 1652082, 1652083, 1652086, 1652088, 1652089, 1652091, 1652092, 1652093, 165-2661, pulsetrac, pulse trac, shock pod, 1652660, 1652662, 165-2660J1, 165-2661J1, 165-2662J1, 165-2666, 165-2666J1, 165-2667, 1652660J1, 1652661J1, 1652662J1, 1652666, 1652666J1, 1652667, 170-2503, 170-2503EDU, 170-2504, 170-2505, 1702503, 1702503EDU, 1702504, 1702505 1359 High Electro-transformation Efficiencies Obtained With DNA From Ligation Mixtures 1359 /webroot/web/pdf/lsr/literature/Bulletin_1359.pdf Literature PDF Articles_and_Whitepapers /webroot/web/images/general/icons/icon_pdf.gif No High Electro-transformation Efficiencies Obtained With DNA From Ligation Mixtures 1359 lit1359, bulletin 1359, e. coli, ligation, efficiency, cloning, pulser, electroporation, transformation, 165-2101, 165-2102, 165-2103, 165-2104, 1652101, 1652102, 1652103, 1652104, transfection, trans-fection, genepulser, gene pulser, electroporator, 165-2660, 165-2662, condition, protocol, shockpod, 1652661, protocols, 1652081, 1652082, 1652083, 1652086, 1652088, 1652089, 1652091, 1652092, 1652093, 165-2661, pulsetrac, pulse trac, shock pod, 1652660, 1652662, 165-2660J1, 165-2661J1, 165-2662J1, 165-2666, 165-2666J1, 165-2667, 1652660J1, 1652661J1, 1652662J1, 1652666, 1652666J1, 1652667, 170-2503, 170-2503EDU, 170-2504, 170-2505, 1702503, 1702503EDU, 1702504, 1702505 1358 Efficient Cloning and Electro-transformation of Large Eukaryotic DNA Fragments 1358 /webroot/web/pdf/lsr/literature/Bulletin_1358.pdf Literature PDF Articles_and_Whitepapers /webroot/web/images/general/icons/icon_pdf.gif No Efficient Cloning and Electro-transformation of Large Eukaryotic DNA Fragments 1358 lit1358, bulletin 1358, cloning, large, eukaryotic, dna, fragment, fragments, gene transfer, transfection, gene pulser, electroporation, 165-2660, 165-2661, 165-2662, 165-2666, 165-2667, 165-2668, 165-2669, 1652660, 1652661, 1652662, 1652666, 1652667, 1652668, 1652669, trans-fection, transformation, genepulser, electroporator, condition, protocol, shockpod, efficiency, protocols, 1652081, 1652082, 1652083, 1652086, 1652088, 1652089, 1652091, 1652092, 1652093, pulsetrac, pulse trac, shock pod, 165-2660J1, 165-2661J1, 165-2662J1, 165-2666J1, 1652660J1, 1652661J1, 1652662J1, 1652666J1, 170-2503, 170-2503EDU, 170-2504, 170-2505, 1702503, 1702503EDU, 1702504, 1702505 1353 Electro-transformation of <em>E. coli</em> With M13 DNA 1353 /webroot/web/pdf/lsr/literature/Bulletin_1353.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Electro-transformation of <em>E. coli</em> With M13 DNA 1353 lit1353, bulletin 1353, m13 vectors, ecoli, e. coli, m13, phage, phagemid, gene pulser, electroporation, transformation, mutagenesis, 165-2660, 165-2661, 165-2662, 165-2666, 165-2667, 165-2668, 165-2669, 1652660, 1652661, 1652662, 1652666, 1652667, 1652668, 1652669, transfection, trans-fection, genepulser, electroporator, condition, protocol, shockpod, efficiency, protocols, 1652081, 1652082, 1652083, 1652086, 1652088, 1652089, 1652091, 1652092, 1652093, pulsetrac, pulse trac, shock pod, 165-2660J1, 165-2661J1, 165-2662J1, 165-2666J1, 1652660J1, 1652661J1, 1652662J1, 1652666J1, 170-2503, 170-2503EDU, 170-2504, 170-2505, 1702503, 1702503EDU, 1702504, 1702505 Life Science Research/Products/Chromatography/Affinity Chromatography and Tag Cleavage Kit Products/Profinity eXact Fusion-Tag System/Profinity eXact Expression Vector Kits and Cloning Products ->MT::9e5bff3d-ab72-4e1d-bc77-68c6368104da##Life Science Research/Products/Transfection/Electroporation/Gene Pulser Xcell Electroporation Systems ->MT::b1a35eb3-d55c-47b3-aaf3-95e4d1d85848##Life Science Education/Products/Equipment and Supplies/PCR Instrumentation/C1000 Thermal Cycler ->MT::dcdab038-4e77-49a8-bcaa-f67e8648550d##Life Science Research/Products/Amplification - PCR/Real-Time PCR Detection Systems/CFX96 Touch Real-Time PCR Detection System ->MTS::LJB1YU15##Life Science Research/Products/Amplification - PCR/PCR Reagents/Core Reagents for PCR/iProof High-Fidelity DNA Polymerase ->MT::693510a6-26f0-4487-ab4a-7cff7222d4ac## Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/MIQE and RDML ->MTS::LUSO8N4EH##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Assay Design and Optimization ->MTS::LUSO7RIVK##Life Science Research/Solutions/Technologies/PCR/Reagents ->MTS::LUSO0V4VY##Life Science Research/Solutions/Technologies/PCR/Analysis ->MTS::LUSO2PESH## Eddie C What is Gene Cloning? 12/20/11 02:35 PM 12/20/21 02:36 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Applications/Genomics N 0 Genomics /en-us/applications-technologies/applications-technologies/introduction-gene-cloning-analysis?ID=LUSNI3MNI

Gene cloning is a common practice in molecular biology labs that is used by researchers to create copies of a particular gene for downstream applications, such as sequencing, mutagenesis, genotyping or heterologous expression of a protein. The traditional technique for gene cloning involves the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element, such as a bacterial plasmid. This technique is commonly used today for isolating long or unstudied genes and protein expression. A more recent technique is the use of polymerase chain reaction (PCR) for amplifying a gene of interest. The advantage of using PCR over traditional gene cloning, as described above, is the decreased time needed for generating a pure sample of the gene of interest. However, gene isolation by PCR can only amplify genes with predetermined sequences. For this reason, many unstudied genes require initial gene cloning and sequencing before PCR can be performed for further analysis.

Related Topics: Gene Expression Analysis, Mutational Analysis, and Epigenetics and Chromatin Structure.

 

DNA Sequencing

DNA sequencing is typically the first step in understanding the genetic makeup of an organism, which helps to:

  • Locate regulatory and gene sequences
  • Compare homologous genes across species
  • Identify mutations

Sequencing uses biochemical methods to determine the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a DNA oligonucleotide. Knowing the sequence of a particular gene will assist in further analysis to understand the function of the gene. PCR is used to amplify the gene of interest before sequencing can be performed. Many biotechnology companies offer sequencing instruments, however, these instruments can be expensive. As a result, many researchers usually perform PCR in-house and then send out their samples to sequencing labs.

 

Site-Directed Mutagenesis

Site-directed mutagenesis is a widely used procedure for the study of the structure and function of proteins by modifying the encoding DNA. By using this method, mutations can be created at any specific site in a gene whose wild-type sequence is already known. Many techniques are available for performing site-directed mutagenesis. A classic method for introducing mutations, either single base pairs or larger insertions, deletions, or substitutions into a DNA sequence, is the Kunkel method.

The first step in any site-directed mutagenesis method is to clone the gene of interest. For the Kunkel method, the cloned plasmid is then transformed into a dut ung mutant of Escherichia coli. This E. coli strain lacks dUTPase and uracil deglycosidase, which will ensure that the plasmid containing the gene of interest will be converted to DNA that lacks Ts and contains Us instead.

The next step is to design a primer that contains the region of the gene which you wish to mutate, along with the mutation you want to introduce. PCR can then be used with the mutated primers to create hybrid plasmids; each plasmid will now contain one strand without the mutation and uracil bases, and another strand with the mutation and lacking uracil.

The final step is to isolate this hybrid plasmid and transform it into a different strain that does contain the uracil-DNA glycosylase (ung) gene. The uracil deglycosidase will destroy the strands that contain uracil, leaving only the strands with your mutation. When the bacteria replicate, the resulting plasmids will contain the mutation on both strands.

 

Genotyping

Genotyping is the process of determining the DNA sequence specific to an individual's genotype. This process can be accomplished by several techniques, such as high resolution melt (HRM) analysis, or any other mutation detection technique. All of these techniques will provide an insight into the individual's genotype, which can help determine specific sequences that can be manipulated and cloned for further analysis.

 

Heterologous Protein Expression

Heterologous protein expression uses gene cloning to express a protein of interest in a self-replicating genetic element, such as a bacterial plasmid. Heterologous expression is used to produce large amounts of a protein of interest for functional and biochemical analyses.

 

Further Reading

Araya-Garay JM et al. (2011). cDNA cloning of a novel gene codifying for the enzyme lycopene β-cyclase from Ficus carica and its expression in Escherichia coli. Appl Microbiol Biotechnol 92, 769–777. PMID: 21792589

Eckert C et al. (2006). DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother 57, 14–23. PMID: 16291869

Handa P and Varshney U (1998). Rapid and reliable site-directed mutagenesis using Kunkel's approach. Indian J Biochem Biophys 35, 63–66. PMID: 9753863

Hanner M et al. (1996). Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc Natl Acad Sci USA 93, 8072–8077. PMID: 8755605

Kerovuo J et al. (1998). Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl Environ Microbiol 64, 2079–2085. PMID: 9603817

Lauretti L et al. (1999). Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate. J Antimicrob Chemother 43, 1584–1590. PMID: 10390207

Morales G et al (2010). Resistance to linezolid is mediated by the cfr gene in the first report of an outbreak of linezolid-resistant Staphylococcus aureus. Clin Infect Dis 50, 821–825. PMID: 20144045

Naldini L et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267. PMID: 8602510

Riordan JR et al. (1999). Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066–1073. PMID: 2475911

 

Related Content

 
Literature
Number Description Download
2249 Examples of Electro-Transfected Eukaryotic and Prokaryotic Cells, Rev A Click to download
1359 High Electro-transformation Efficiencies Obtained With DNA From Ligation Mixtures Click to download
1358 Efficient Cloning and Electro-transformation of Large Eukaryotic DNA Fragments Click to download
1353 Electro-transformation of <em>E. coli</em> With M13 DNA Click to download
 
 
LUSNKO4EH [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [54.158.52.166, 10.232.2.159] [accept] = [text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8] [if-modified-since] = [Fri, 20 Jul 2018 03:17:51 GMT] [seourl] = [/en-us/applications-technologies/applications-technologies/introduction-gene-cloning-analysis] [x-amzn-trace-id] = [Root=1-5ba5f826-b0257ed22f98f3f4b3562b63] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=LUSNKO4EH] [host] = [10.232.0.21:1776] [x-request-uri] = [/en-us/applications-technologies/applications-technologies/introduction-gene-cloning-analysis] [connection] = [Keep-Alive] [accept-encoding] = [gzip] [user-agent] = [CCBot/2.0 (https://commoncrawl.org/faq/)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/applications-technologies/introduction-gene-cloning-analysis LSR LUSNKO4EH Gene Cloning and Analysis Introduction to Gene Cloning and Analysis /webroot/web/html/lsr/solutions/applications/genomics <p>Gene cloning is a common practice in molecular biology labs that is used by researchers to create copies of a particular gene for downstream applications, such as sequencing, mutagenesis, genotyping or heterologous expression of a protein. The traditional technique for gene cloning involves the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element, such as a bacterial plasmid. This technique is commonly used today for isolating long or unstudied genes and protein expression. A more recent technique is the use of <a href="/evportal/destination/solutions?catID=LUSNYI15">polymerase chain reaction (PCR)</a> for amplifying a gene of interest. The advantage of using PCR over traditional gene cloning, as described above, is the decreased time needed for generating a pure sample of the gene of interest. However, gene isolation by PCR can only amplify genes with predetermined sequences. For this reason, many unstudied genes require initial gene cloning and sequencing before PCR can be performed for further analysis.</p> <p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSNINKSY">Gene Expression Analysis,</a> <a href="/evportal/destination/solutions?catID=LUSNLMLPT">Mutational Analysis,</a> and <a href="/evportal/destination/solutions?catID=LUSNMFHYP">Epigenetics and Chromatin Structure.</a></p> DNA Sequencing <p><a name="dna_sequencing"></a>DNA sequencing is typically the first step in understanding the genetic makeup of an organism, which helps to:</p> <ul> <li>Locate regulatory and gene sequences</li> <li>Compare homologous genes across species</li> <li>Identify mutations</li> </ul> <p>Sequencing uses biochemical methods to determine the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a DNA oligonucleotide. Knowing the sequence of a particular gene will assist in further analysis to understand the function of the gene. <a href="/evportal/destination/solutions?catID=LUSOBDHYP">PCR</a> is used to amplify the gene of interest before sequencing can be performed. Many biotechnology companies offer sequencing instruments, however, these instruments can be expensive. As a result, many researchers usually perform PCR in-house and then send out their samples to sequencing labs.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Site-Directed Mutagenesis <p><a name="site_directed"></a>Site-directed mutagenesis is a widely used procedure for the study of the structure and function of proteins by modifying the encoding DNA. By using this method, mutations can be created at any specific site in a gene whose wild-type sequence is already known. Many techniques are available for performing site-directed mutagenesis. A classic method for introducing mutations, either single base pairs or larger insertions, deletions, or substitutions into a DNA sequence, is the Kunkel method.</p> <p>The first step in any site-directed mutagenesis method is to clone the gene of interest. For the Kunkel method, the cloned plasmid is then transformed into a dut ung mutant of <em>Escherichia coli</em>. This <em>E. coli</em> strain lacks dUTPase and uracil deglycosidase, which will ensure that the plasmid containing the gene of interest will be converted to DNA that lacks Ts and contains Us instead.</p> <p>The next step is to design a primer that contains the region of the gene which you wish to mutate, along with the mutation you want to introduce. PCR can then be used with the mutated primers to create hybrid plasmids; each plasmid will now contain one strand without the mutation and uracil bases, and another strand with the mutation and lacking uracil.</p> <p>The final step is to isolate this hybrid plasmid and transform it into a different strain that does contain the uracil-DNA glycosylase (ung) gene. The uracil deglycosidase will destroy the strands that contain uracil, leaving only the strands with your mutation. When the bacteria replicate, the resulting plasmids will contain the mutation on both strands.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Genotyping <p>Genotyping is the process of determining the DNA sequence specific to an individual's genotype. This process can be accomplished by several techniques, such as high resolution melt (<a href="/evportal/destination/solutions?catID=LUSOIH97Q">HRM</a>) analysis, or any other <a href="/evportal/destination/solutions?catID=LUSNLMLPT">mutation detection technique</a>. All of these techniques will provide an insight into the individual's genotype, which can help determine specific sequences that can be manipulated and cloned for further analysis.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Heterologous Protein Expression <p>Heterologous protein expression uses gene cloning to express a protein of interest in a self-replicating genetic element, such as a bacterial plasmid. Heterologous expression is used to produce large amounts of a protein of interest for functional and biochemical analyses.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Further Reading <p>Araya-Garay JM et al. (2011). cDNA cloning of a novel gene codifying for the enzyme lycopene &beta;-cyclase from <em>Ficus carica</em> and its expression in <em>Escherichia coli</em>. Appl Microbiol Biotechnol 92, 769&ndash;777. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/21792589" target="_blank">21792589</a></p> <p>Eckert C et al. (2006). DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother 57, 14&ndash;23. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/16291869" target="_blank">16291869</a></p> <p>Handa P and Varshney U (1998). Rapid and reliable site-directed mutagenesis using Kunkel's approach. Indian J Biochem Biophys 35, 63&ndash;66. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/9753863" target="_blank">9753863</a></p> <p>Hanner M et al. (1996). Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc Natl Acad Sci USA 93, 8072&ndash;8077. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/8755605" target="_blank">8755605</a></p> <p>Kerovuo J et al. (1998). Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from <em>Bacillus subtilis</em>. Appl Environ Microbiol 64, 2079&ndash;2085. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/9603817" target="_blank">9603817</a><br /> <br /> Lauretti L et al. (1999). Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a <em>Pseudomonas aeruginosa</em> clinical isolate. J Antimicrob Chemother 43, 1584&ndash;1590. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/10390207" target="_blank">10390207</a></p> <p>Morales G et al (2010). Resistance to linezolid is mediated by the cfr gene in the first report of an outbreak of linezolid-resistant <em>Staphylococcus aureus</em>. Clin Infect Dis 50, 821&ndash;825. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/20144045" target="_blank">20144045</a></p> <p>Naldini L et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263&ndash;267. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/8602510" target="_blank">8602510</a></p> <p>Riordan JR et al. (1999). Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066&ndash;1073. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/2475911" target="_blank">2475911</a></p> <div class="top"><a href="#helptop">Back to Top</a></div> 2249 1359 1358 1353 Life Science Research/Products/Chromatography/Affinity Chromatography and Tag Cleavage Kit Products/Profinity eXact Fusion-Tag System/Profinity eXact Expression Vector Kits and Cloning Products ->MT::9e5bff3d-ab72-4e1d-bc77-68c6368104da##Life Science Research/Products/Transfection/Electroporation/Gene Pulser Xcell Electroporation Systems ->MT::b1a35eb3-d55c-47b3-aaf3-95e4d1d85848##Life Science Education/Products/Equipment and Supplies/PCR Instrumentation/C1000 Thermal Cycler ->MT::dcdab038-4e77-49a8-bcaa-f67e8648550d##Life Science Research/Products/Amplification - PCR/Real-Time PCR Detection Systems/CFX96 Touch Real-Time PCR Detection System ->MTS::LJB1YU15##Life Science Research/Products/Amplification - PCR/PCR Reagents/Core Reagents for PCR/iProof High-Fidelity DNA Polymerase ->MT::693510a6-26f0-4487-ab4a-7cff7222d4ac## Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/MIQE and RDML ->MTS::LUSO8N4EH##Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR/Assay Design and Optimization ->MTS::LUSO7RIVK##Life Science Research/Solutions/Technologies/PCR/Reagents ->MTS::LUSO0V4VY##Life Science Research/Solutions/Technologies/PCR/Analysis ->MTS::LUSO2PESH## Eddie C What is Gene Cloning? 12/20/11 02:35 PM 12/20/21 02:36 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Applications/Genomics N 0
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