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Chapter 19

Textbook and Class Notes Collaborated - Unit 3 - Chapter 19 Bio 1A03

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McMaster University
Xudong Zhu

Bio 1A03 Unit Three: Gene Structure and Expression Chapter 19: Analyzing and Engineering Genes Key Concepts  The discovering of restriction endonucleases that cut DNA at specific locations, along with ligases that piece DNA segments back together, gave biologists the ability to move gene from one location to another  To analyze a gene, biologists have to obtain many identical copies of it o Can be done by inserting the gee into a bacterial cell that copies the gene as it grows, or by performing a polymerase chain reaction (PCR)  Once researchers have many identical copies of a gene, its sequence of bases can be determined by dideoxy sequencing  Researchers are attempting to insert genes into humans to cure genetic diseases o Efforts to insert genes into plants to provide them with novel traits, such as the ability to resist insect attacks, have been much more successful 19.1 Using Recombinant DNA Technology  Genetic Engineering – manipulation of DNA sequences in organisms  Recombinant DNA technology –techniques used to engineered genes  Goals of Genetic Engineering o Improve our understanding of how genes work o Advance biotechnology – the manipulation of organisms to create products or cure diseases Effort to Cure Pituitary Dwarfism  Pituitary Gland – structure at the base of the mammalian brain that produces several important molecules – including a protein that stimulates growth  Pituitary Dwarfism results from the lack of production of growth hormone, encoded by the GH1 gene  Pituitary dwarfism type I is an autosomal recessive trait o Affected individuals have two copies of the defective allele  Humans affected by pituitary dwarfism grow slowly, reaching a maximum adult height of about 4 feet Why Did Early Efforts to Treat the Disease Fail?  Early trials showed that people with pituitary dwarfism could be treated successfully with growth hormone therapy, but only if the protein came from humans  Growth hormone purified from the pituitary glands of human cadavers is scarce and expensive  Human treatment with growth hormone from cadavers has been banned due to possible contamination with prions – protein particles that have been implicated as the cause of various neurodegenerative disorders Producing a Safe Supply of Growth Hormone  The recombinant DNA strategy for producing human growth hormone involved in cloning the human gene, introducing the gene into bacteria and having the recombinant cells produce the hormone Using Reverse Transcriptase to Produce cDNAs  Reverse transcriptase can synthesize DNA from an RNA template o Reverse transcriptase synthesizes a single stranded cDNA Bio 1A03 o Researchers add a primer to single-stranded cDNA and use DNA polymerase to synthesize the complementary strand of cDNA – although reverse transcriptase is capable of synthesizing the complementary strand for cDNA  Researchers use reverse transcriptase to make complementary DNA (cDNA) from mRNA isolated from pituitary cells o cDNA – DNA made from an RNA template  They then use DNA cloning – the process of producing many identical copies of a gene – to copy the cDNAs for analysis to determine which encoded the growth hormone protein How Are Plasmids Used in Cloning?  Plasmids – small circular DNA molecules often found in bacteria o They replicate independently of the chromosome o Can serve as a vector – a vehicle for transferring recombinant genes to a new host  If a recombinant plasmid can be inserted into a bacterial or yeast cell, the foreign DNA will be copied and transmitted to new cells as the host cell grows and divides o Plasmids can be used to produce millions of identical copies of specified genes  Can harvest the recombinant gene by breaking the bacteria open, isolating all of the DNA and then separating the plasmids from the main chromosomes Cutting and Pasting DNA  Restriction endonucleases – enzymes that cut DNA at recognition sites (specific palindromic sequences)  First step – cut the plasmid and the cDNA with the same restriction endonuclease  Restriction endonucleases often make staggered cuts in the DNA – resulting in sticky ends o Sticky ends – single-stranded ends that are complementary to each other Transformation  Plasmid vectors can be introduced into bacteria by transformation – the process of taking up DNA from the environment and incorporating it into their genome Bio 1A03  To transform bacterial cells with a plasmid o Researchers increase the permeability of the cells plasma membranes using a specific chemical treatment or an electrical shock o Typically, just a single plasmid enters the cell during this treatment o Resulting cells are spread out on plates o The cells are then allowed to grow into colonies How to Clone a Gene of Your Interest From a Genome Producing a cDNA Library  cDNA library – a collection of bacterial cells, each containing a vector with one cDNA  A genomic library is made up of cloned DNA fragments representing an entire genome  DNA libraries are important because they give researchers a way to store information from a particular cell type or genome in an accessible form How is a cDNA Library Screened?  DNA probe – single-stranded fragment of a known gene that binds a complementary sequence in the sample of DNA being analyzed o By binding to the target sequence, it distinguishes it from the other DNA fragments  A DNA probe must be labeled so it can be found after it has bound the target sequence  The growth hormone researchers inferred the approximate sequence for the GH1 gene from the amino acid sequence of human growth hormone  They constructed a probe based on this inferred sequence and radioactively labeled it  They then used this probe to screen a cDNA library for the plasmid containing the GH1 cDNA Bio 1A03 Mass Producing Growth Hormone  Once researchers found the human growth hormone cDNA, they cloned it in a plasmid under the control of a bacterial promoter  The transformed E. coli cells produced human growth hormone that could be isolated and purified in large quantities Ethical Concerns Over Recombinant Growth Hormone  The increased supply of growth hormone led to its use to treat children who were short but did not suffer from pituitary dwarfism  The US Food and Drug Administration has now approved use of the hormone only for children projected to reach adult heights of less than 5’3 for males and 4’11 for females 19. 2 Another Approach to Cloning  The polymerase chain reaction (PCR) is an in vitro DNA synthesis reaction in which a specific DNA sequence is replicated over and over again o This technique generated many identical copies of a particular DNA sequence The Polymerase Chain Reaction  PCR is possible only when DNA sequence information surrounding the gene of interest is available, because PCR requires primers that match sequences on either side of the gene  One primer is complementary to a sequence on one strand upstream of the target DNA and the other primer is commentary to a sequence on the other strand downstream of the target  The primers will bind to single-stranded target DNA  PCR Reaction contains o A DNA template o Two primers that bracket the reg
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