Chapter 20 Recombinant DNA Technology. Copyright 2009 Pearson Education, Inc.

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1 Chapter 20 Recombinant DNA Technology Copyright 2009 Pearson Education, Inc.

2 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors Recombinant DNA refers to the joining of DNA molecules, usually from different biological sources, that are not found together in nature 2012 Pearson Education, Inc.

3 The basic procedure for producing recombinant DNA involves generating specific DNA fragments using restriction enzymes joining these fragments with a vector transferring the recombinant DNA molecule to a host cell to produce many copies that can be recovered from the host cell The recovered copies of a recombinant DNA molecule are referred to as clones and can be used to study the structure and orientation of the DNA. Recombinant DNA technology is used to isolate, replicate, and analyze genes Pearson Education, Inc.

4 A restriction enzyme binds to DNA at a specific recognition sequence and cleaves the DNA to produce restriction fragments. Most recognition sequences are palindromic, and restriction enzymes often cleave these sequences in an offset manner. DNA ligase joins restriction fragments covalently to produce intact DNA molecules.

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7 Vectors are carrier DNA molecules that can replicate cloned DNA fragments in a host cell. Vectors must be able to replicate independently and should have several restriction enzyme sites to allow insertion of a DNA fragment. Vectors should carry a selectable gene marker to distinguish host cells that have taken them up from those that have not Pearson Education, Inc.

8 Expression vectors are engineered to express a gene of interest to produce large quantities of the encoded protein A plasmid is an extrachromosomal double-stranded DNA molecule that replicates independently from the chromosomes within bacterial cells. Plasmids used for DNA cloning usually have been engineered to contain: a number of convenient restriction sites a marker gene to select for its presence in the host cell 2012 Pearson Education, Inc.

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13 The central third of lambda (λ) phage vectors can be replaced with foreign DNA without affecting the ability to infect cells and replicate. Lambda vectors can carry up to 20 kb of cloned DNA 2012 Pearson Education, Inc.

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15 Cosmid vectors are created by combining parts of λ phage and parts of plasmids. Cosmids contain the cos sites of lambda, which are necessary for packaging of phage DNA into phage particles. Once inside the bacterial cell, cosmids replicate as plasmids. Cosmids can carry almost 50 kb of inserted DNA Pearson Education, Inc.

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17 Bacterial artificial chromosomes (BACs) are based on F factor and can carry up to 300 kb of inserted DNA Pearson Education, Inc.

18 Yeast Cells Are Used as Eukaryotic Hosts for Cloning Yeast is widely used as a host for DNA cloning because: it can be grown easily its genetics have been studied intensively its genome has been sequenced it can posttranslationally modify eukaryotic proteins it is considered to be safe

19 Yeast artificial chromosomes can contain kb of inserted DNA.

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21 Plant and Animal Cells Can Be Used As Host Cells For Cloning Plant Cell Hosts: Agrobacterium tumefaciens can be used to transform plant cells with T- DNA containing foreign DNA 2012 Pearson Education, Inc.

22 From Agrobacterium tumifaciens 2012 Pearson Education, Inc.

23 The T-DNA and insert integrate into the plant genome. The plant cells can be grown in tissue culture and eventually regenerate a mature plant carrying a foreign gene. DNA can be transferred to mammalian cells by several methods, including endocytosis and encapsulation in liposomes followed by fusion with cell membranes.

24 A variety of different human cell types can be grown in culture and used to express genes and proteins. These lines can be subjected to various approaches for gene or protein functional analysis, including drug testing for effectiveness at blocking or influencing a particular recombinant protein being expressed. Transgenic mice can be produced by transferring YACs by microinjection into the nucleus of a mouse oocyte Pearson Education, Inc.

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26 20.2 DNA Libraries Are Collections of Cloned Sequences DNA libraries represent a collection of cloned DNA samples derived from a single source that could be a particular tissue type, cell type, or single individual. A genomic library contains at least one copy of all the sequences in the genome of interest. Genomic libraries are constructed by cutting genomic DNA with a restriction enzyme and ligating the fragments into vectors, which are chosen depending on the size of the genome 2012 Pearson Education, Inc.

27 cdna libraries contains complementary DNA copies made from the mrnas present in a cell population and represents the genes that are transcriptionally active. A cdna library is prepared by isolating mrna from cells synthesizing the complementary DNA using reverse transcriptase cloning the cdna molecules into a vector

28 Specific Clones Can Be Recovered from a Library Probes complementary to part of a gene are used to screen a library to recover clones of a specific gene. To screen a plasmid library, clones from the library are grown on agar plates to produce colonies. The colonies are screened by transferring bacterial colonies from the plate to a filter and hybridizing the filter with a nucleic acid probe to the DNA sequence of interest. The colony corresponding to the one the probe identified on the filter is identified and recovered.

29 2012 Pearson Education, Inc. Producing cdna from mrna

30 2012 Pearson Education, Inc. Screening a library constructed using a plasmid vector to recover a specific gene

31 20.3 PCR Is a Powerful Technique for Copying DNA The Polymerase Chain Reaction Makes DNA Copies without Host Cells 2012 Pearson Education, Inc.

32 PCR copies a specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities. PCR requires two primers, one complementary to the 3' end of one strand to be amplified and one complementary to the 3' end of the other strand. The primers anneal to denatured DNA, and complementary strands are synthesized by a heat-stable DNA polymerase. The three steps of PCR (denaturation, primer annealing, and extension) are repeated over and over using a thermocycler to amplify the DNA exponentially.

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34 Reverse transcription PCR (RT-PCR) is used to study gene expression by studying mrna production by cells or tissues Quantitative real-time PCR (qpcr) or real-time PCR allows researchers to quantify amplification reactions as they occur in real time. The procedure uses an SYBR green dye and TaqMan probes, which contain two dyes 2012 Pearson Education, Inc.

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36 2012 Pearson Education, Inc Molecular Techniques for Analyzing DNA

37 20.4 Molecular Techniques for Analyzing DNA A restriction map establishes the number and order of restriction sites and the distance between restriction sites on a cloned DNA segment. It provides information about the length of the cloned insert and the location of restriction sites within the clone Pearson Education, Inc.

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39 2012 Pearson Education, Inc. Constructing a restriction map

40 A Southern blot is used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments from restriction digest. Southern blots can also be used to determine whether a clone contains all or part of a gene. ascertain the size and sequence organization of a gene or DNA sequence of interest.

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44 Northern blot analysis is used to determine whether a gene is actively being expressed in a given cell or tissue. Used to study patterns of gene expression in embryonic tissues, cancer, and genetic disorders Pearson Education, Inc.

45 20.5 DNA Sequencing Is the Ultimate Way to Characterize DNA Structure at the Molecular Level The most common method of DNA sequencing is dideoxy chain termination sequencing.

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47 2012 Pearson Education, Inc. A A T G C G T T 5 -TTCGTGAA etc

48 Large-scale genome sequencing is automated and uses fluorescent dye-labeled dideoxynucleotides Pearson Education, Inc.

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