BIOL 3451 1st Edition Lecture 26 Outline of Last Lecture I. 19.8 Environmental Agents Contribute to Human CancersII. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning VectorsIII. 20.2 DNA Libraries Are Collections of Cloned SequencesOutline of Current Lecture I. 20.2 DNA Libraries Are Collections of Cloned SequencesII. 20.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNAIII. 20.4 Molecular Techniques for Analyzing DNAIV. 20.5 DNA Sequencing Is the Ultimate Way to Characterize DNA Structure at the Molecular Level Current LectureI. 20.2 DNA Libraries Are Collections of Cloned Sequenceso 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 genomeo YACs were commonly used to accommodate large sizes of DNA necessary to span the 3 billion ppb of DNA in the human genomeo Whole-genome shotgun cloning approaches and next-generation sequencing are replacing traditional genomic libraries- Today, we don’t even use cloning techniques to get genomeo All DNA fragments in a genomic sample are sequenced without the need to insert DNA fragments into vectors and cloning them in host cellsThese 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.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 transcriptase3. 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 amounto 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 colonies1. 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. 20.73. The colony corresponding to the one the probe identified on the filter is identified and recovereda. Phages are sometimes used because they are more useful sincethey pick up larger pieces4. A phage library screened by plaque hybridizationa. Allows scientists to clone DNA and then identify individual genes in the libraryo 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 identifiedII. 20.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNAo 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- 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 exponentially1. DNA strand is doubled in each cycle, and new strands along with the old strand serve as templates in next cycleo 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 DNAo 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 disorderso PCR useful with- Single cell samples- Fossils- Crime scene where single hair or a little saliva was foundo PCR used to enforce worldwide ban on the sale of certain whale products and determination of pedigree background of purebred dogso Reverse Transcription PCR (RT-PCR): used to study gene expression by studying mRNA production by cells or tissues- Good for viral infectionso 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?III. 20.4 Molecular Techniques for Analyzing DNAo 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- 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 wholething, 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)o Southern Blot: used to identify which clones in a