Ebook Introduction to genetic analysis (9th edition): Part 2
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Ebook Introduction to genetic analysis (9th edition): Part 2
GENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2ow is DNA amplified without cloning?•How is amplified DNA used in genetics?•How are DNA technologies applied to medicine?OUTLINE11.1Generating recombinant DNA molecules11.2DNA amplification in vitro: the polymerase chain reaction11.3Zeroing in on the gene for alkaptonuria: another case study11.4Dete Ebook Introduction to genetic analysis (9th edition): Part 2cting human disease alleles: molecular genetic diagnostics11.5Genetic engineeringInjection of foreign DNA into an animal cell. The microneedle used foEbook Introduction to genetic analysis (9th edition): Part 2
r injection is shown at right and a cell-holding pipette at left [Copyright M. Baiet.'RaptwPhotc Raaeaichtfs, Inc }341342Chapter 11 • Gene Isolation aGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2 DNA region) from the genome and amplify it toobtain a working amount to study. DNA technology is a term that describes the collective techniques for obtaining. amplifying, and manipulating specific DNA fragments. Since the mid-1970s, the development of DNA technology has revolutionized the study of Ebook Introduction to genetic analysis (9th edition): Part 2 biology, opening many areas of research to molecular investigation. Genetic engineering, the application of DNA technology to specific biological, meEbook Introduction to genetic analysis (9th edition): Part 2
dical, or agriculturalproblems, is now a well-established branch of technology. Genomics is the ultimate extension of the technology to the global anaGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2 DNA segments be isolated? That task initially might seem like finding a needle in a haystack. A crucial insight was that researchers could create the large samples of DNA that they needed by tricking the DNA replication machinery to replicate the DNA segment in question. Such replication could be d Ebook Introduction to genetic analysis (9th edition): Part 2one either within live bacterial cells (in vivo) or in a test tube (in vitro).CHAPTER OVERVIEW FigureChromosomeGene of rterestQ Enzymes that bind to DEbook Introduction to genetic analysis (9th edition): Part 2
NA EZ> Primer for DNA polymerizationFigure 11-1 How to amplify an interesting gene. Two methods are (a) in vivo, by tricking the replication machineryGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2olecular biology: the ability of specific proteins to bind to DNA (the proteins shown in yellow) and the ability of complementary Single-Stranded nucleic acid segments to hybridize together (the primer used in the test-tube method).34311.1 Generating recombinant ONA moleculesIn the in vivo approach Ebook Introduction to genetic analysis (9th edition): Part 2(Figure 11 - la), the investigator begins with a sample of DNA molecules containing the gene of interest. This sample is called the donor DNA and mostEbook Introduction to genetic analysis (9th edition): Part 2
often it is an entire genome. Fragments of the donor DNA are inserted into nonessential •’accessory" chromosomes (such as plasmids or modified bacterGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2ie cut up. by using enzymes called restriction endonucleases as molecular "scissors." These enzymes are a class of DNA-binding proteins that bind to the DNA and cut the sugar-phosphate backbone of each of the two strands of the double helix at a specific sequence. They cut long chromosome-sized DNA Ebook Introduction to genetic analysis (9th edition): Part 2molecules into hundreds or thousands of fragments of more manageable size. Next, each fragment is fused with a cut vector chromosome to form recombinaEbook Introduction to genetic analysis (9th edition): Part 2
nt DN’A molecules. Union with the vector DNA typically depends on short terminal single strands produced by the restriction enzymes. They bond to compGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2NA.) The recombinant DNAs are inserted into bacterial cells, and generally only one recombinant molecule is taken up by each cell. Because the accessory chromosome is normally amplified by replication, the recombinant molecule is similarly amplified during the growth and division of the bacterial ce Ebook Introduction to genetic analysis (9th edition): Part 2ll in which the chromosome resides. This process results in a clone of identical cells, each containing the recombinant DNA molecule, and so this techEbook Introduction to genetic analysis (9th edition): Part 2
nique of amplification is called DNA cloning. The next stage IS finding the rare clone containing the DNA of interest.In the in vitro approach (FigureGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2interest by the complementary binding of specific short primers to the ends of that sequence. These primers then guide the replication process, which cycles exponentially, resulting in a large sample of copies of the gene of interest.We will see repeatedly that DNA technology depends on two basic fo Ebook Introduction to genetic analysis (9th edition): Part 2undations of molecular biolog}' research:•The ability of specific proteins to recognize and bind to specific base sequences, within the DNA double helEbook Introduction to genetic analysis (9th edition): Part 2
ix (examples are shown in yellow in Figure 11-1).•The ability of complementary single-stranded DNA or RNA sequences to spontaneously unite to form douGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2of uses to which we put amplified DNA. These uses range from routine gene isolation for basic biological research to gene-based therapy of human disease.11.1 Generating recombinant DNA molecules To illustrate how recombinant DNA is made, let s consider the cloning of the gene for human insulin, a pr Ebook Introduction to genetic analysis (9th edition): Part 2otein hormone used in the treatment of diabetes. Diabetes is a disease in which blood sugar levels are abnormally high either because the body does noEbook Introduction to genetic analysis (9th edition): Part 2
t produce enough insulin (type I diabetes) or because cells are unable to respond to insulin I type II diabetes). In mild forms of type I. diabetes caGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•Ho Ebook Introduction to genetic analysis (9th edition): Part 2urce of insulin protein. The protein was harvested from the pancreases of animals slaughtered m meat-packing plants and purified at large scale to eliminate the majority of proteins and other contaminants in the pancreas extracts. Then, in 19S2. the first recombinant human insulin came on the drug m Ebook Introduction to genetic analysis (9th edition): Part 2arket. Human insulin could be made purer, at lower cost, and on an industrial scale because it was produced in bacteria by recombinant DNA techniques.Ebook Introduction to genetic analysis (9th edition): Part 2
The recombinant insulin IS a higher proportion of the proteins in the bacterial cell: hence the protein purification is much easier. We shall follow GENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•HoGENE ISOLATION AND MANIPULATIONKEY QUESTIONS•How is a gene isolated and amplified by cloning?•How are specific DNAs or RNAs identified in mixtures?•HoGọi ngay
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