UNT BIOL 3451 - Recombinant DNA and Technology (5 pages)

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Recombinant DNA and Technology

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Recombinant DNA and Technology


Looking at genomic libraries, PCR, and other recombinant DNA techniques.

Lecture number:
Lecture Note
University of North Texas
Biol 3451 - Genetics
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BIOL 3451 1st Edition Lecture 26 Outline of Last Lecture I II III 19 8 Environmental Agents Contribute to Human Cancers 20 1 Recombinant DNA Technology Began with Two Key Tools Restriction Enzymes and DNA Cloning Vectors 20 2 DNA Libraries Are Collections of Cloned Sequences Outline of Current Lecture I 20 2 DNA Libraries Are Collections of Cloned Sequences II 20 3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA III 20 4 Molecular Techniques for Analyzing DNA IV 20 5 DNA Sequencing Is the Ultimate Way to Characterize DNA Structure at the Molecular Level Current Lecture I 20 2 DNA Libraries Are Collections of Cloned Sequences o A genomic library contains at least one copy of all the sequences in the genome of interest 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 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 need to be large to get all of the genome o YACs were commonly used to accommodate large sizes of DNA necessary to span the 3 billion ppb of DNA in the human genome o Whole genome shotgun cloning approaches and next generation sequencing are replacing traditional genomic libraries Today we don t even use cloning techniques to get genome o All DNA fragments in a genomic sample are sequenced without the need to insert DNA fragments into vectors and cloning them in host cells These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute II o Complementary DNA cDNA libraries contains complementary DNA copies made from the mRNAs present in a cell population and represents the genes that are transcriptionally active at the time the cells were collected for mRNA isolation Prepared by 1 isolating mRNA from cells take advantage of poly A tail 2 synthesizing the complementary DNA using reverse transcriptase 3 cloning the cDNA molecules into a vector Fig 20 6 Provides an instant catalog of all the genes active in a cell at a specific time valuable tools for scientists isolating and studying genes in a particular tissue o Reverse transcriptase PCR RT PCR can be used to generate cDNA from mRNA RNA dependent by 1 making a single stranded cDNA copy of the mRNAs using reverse transcriptase T2 using PCR to copy the single stranded DNA into double stranded DNA Easy to make a lot of copies from a very small amount o Library screening used to sort through a library and isolate specific genes of interest To screen a plasmid library clones nucleic acid sequence of what you want from the library are grown on agar plates to produce colonies 1 Transfer bacterial colonies from the plate to a filter and hybridizing the filter with a nucleic acid probe to the DNA sequence of interest in a pattern that matches the pattern on the plates 2 Fig 20 7 3 The colony corresponding to the one the probe identified on the filter is identified and recovered a Phages are sometimes used because they are more useful since they pick up larger pieces 4 A phage library screened by plaque hybridization a Allows scientists to clone DNA and then identify individual genes in the library o Probes used to screen a library to recover clones of a specific gene A probe is any DNA or RNA sequence that is complementary to the target gene of sequence to be identified 20 3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA o Polymerase Chain Reaction PCR copies specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities Rapid way of DNA cloning that eliminates need for using host cells to clone Now easier to make libraries III Ds DNA to be amplified cloned is put in tube with DNA Taq polymerase Mg2 and four deoxyribonucleoside triphosphates Requires two oligonucleotide primers short single stranded sequences one complementary to 5 and the other complementary to 3 Primers anneal to denature DNA fig 20 8 3 steps of PCR denature primer annealing and extension repeated over and over using thermocycler to amplify DNA exponentially 1 DNA strand is doubled in each cycle and new strands along with the old strand serve as templates in next cycle o Limitations of PCR Some info about nucleotide sequence of the target DNA must be known in order to synthesize primer Minor contamination can cause problems Can t amplify long segments of DNA o PCR is useful because Allows screening of mutations involved in genetic disorders Location and nature of mutation can be determined quickly Allele specific probes for genetic testing can be synthesized thus PCR is important for diagnosing genetic disorders o PCR useful with Single cell samples Fossils Crime scene where single hair or a little saliva was found o PCR used to enforce worldwide ban on the sale of certain whale products and determination of pedigree background of purebred dogs o Reverse Transcription PCR RT PCR used to study gene expression by studying mRNA production by cells or tissues Good for viral infections o Quantitative real time PCR qPCR allows for researchers to quantify amplification reactions as they occur in real time Fig 20 9 The procedure uses an SYBR green dye and TaqMan probes which contain two dyes Measure level of gene expression in one step measures how fast you get production of primers 1 Gene turned on If so at what level 20 4 Molecular Techniques for Analyzing DNA o Restriction map used to be like linkage maps but they were not additive distances only relative establishes number and order of restriction sites and the distance between restriction sites on a cloned DNA segment o o o o o Provides info about the length of the cloned insert and the location of restriction sites within the clone Not really used very much anymore now we would just sequence the whole thing and have the computer tell us where it s at Created by cutting DNA with different restriction enzymes and separating the DNA fragments by gel electrophoresis which separates fragments by size The smallest fragments move farthest in the gel These fragments can be visualized when stained with ethidium bromide and illuminated by UV light Figure 20 10 Southern Blot used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments from restriction digest Use paper

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