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Unformatted text preview: Deconstructing the Genome
DNA at High Resolution Chapter 9 I. II. Cutting the DNA: restriction enzymes serve as molecular scissors A. restriction enzymes (RE) fragment the genome at specific sites 0 B. different RE produce fragments of different lengths 1. size of the recognition site is the primary determinant of fragment length 2. timing of exposure to a RE helps determine fragment size C. Different RE produce different numbers of fragments from the same size genome Purification and amplification of fragments for storage and analysis A. Cloning step 1: ligation of fragments to cloning vectors creates recombinant DNA molecules 1. sticky ends facilitate recombinant DNA fabrication 2. there are several types of vectors B. Cloning step 2: recombinant vector: insert vector to host cells, which copy the recombinants along with their own chromosomes 1. transformation: vectors carry insert DNA into cells 2. how do you know which cells have been transformed? 3. how do you know whether the plasmids inside those cells contain an 4. amplification of the recombinant DNA molecule: the actual cloning C. Cloned DNA is purified by various means that separate recombinant plasmid from host DNA, then insert from vector 1. purify recombinant plasmid from host DNA 2. purify inserts from plasmids D. Libraries are collections of cloned fragments 1. genomic libraries are random collections of DNA fragments from organism 2. cDNA libraries carry information from the RNA transcripts insert? III. IV. Identifying and isolating clones of interest 0 A. Screening with DNA probes: hybridization to complementary sequences picks out fragments of interest 1. what is a DNA probe? 2. how to obtain a DNA probe 3. screening with DNA probes 4. requirements and limitations of hybridization probes B. Screening through expression: genes cloned in specialized vectors produce proteins that light up with specific antibodies Characterizing cloned fragments by their size, position and sequence A. Electrophoresis (EP) distinguishes DNA fragments by size; many types of EP B. Restriction maps provide a rough roadmap of a clone C. Hybridization can also serve as a tool of characterization D. Sequencing provides the highest resolution of a cloned fragment; its nucleotide seq. 1. sequencing: the general procedure 2. two strategies for sequencing a clone: directed and shotgun E. Computer analysis of DNA sequences can identify significant genetic motifs as well as resemblances to previously determined sequences F. Polymerase chain reaction (PCR) can amplify a small amount of DNA into a tremendous amount for further analysis 1. how PCR achieves the exponential accumulation of target DNA 2. PCR products can be analyzed and utilized just like cloned restriction fragments 3. the many uses of PCR V. diseases Understanding the genes for hemoglobin: a comprehensive example A. Genes occur in 2 clusters on 2 separate chromosomes B. A variety of mutations accounts for the diverse symptoms of globin-related 0 C. The - and -globin loci house multigene families reflecting key evolutionary events Expression vectors produce large amounts of specific polypeptide 0 Vector contains promotor and regulatory sequences. Vectors transformed into bacteria or yeast.
cDNA = DNA copy made from RNA and contains no introns; can be inserted into vectors easily Screening an expression library Expression library entire cDNA library Probe library with fluorescently labeled antibody that binds to protein product of gene. Hybridization is used to identify similar DNA sequences Prepare library. Distribute library's clones on petri dish. Transfer clones to nitrocellulose disk. Previous cloned DNA PCR fragment Oligonucleotide Expose probe to clones on nitrocellulose. Determine location of matching clone by autoradiography or fluorescence. Prepare probe. Screen library. Gel electrophoresis and hybridization to map DNA fragments Southern blot
Cut whole genomic DNA with restriction enzyme. Separate DNA fragments by electrophoresis. Blot fragmented DNA to a filter. Hybridize to DNA probe. Observe matched bands by autoradiography or fluorescence. 0 Stain with Ethidium bromide Blot is removed Washed, and exposed to X-ray film Polymerase chain reaction to rapidly isolate DNA fragments PCR (polymerase chain reaction) achieved exponential accumulation of target DNA
Based on previously determined DNA sequence, develop short oligonucleotides (~ 20bp) complementary to sequences flanking the target DNA. Oligonucleotides act as primers to copy DNA similar to DNA replication. Polymerase chain reaction to rapidly isolate DNA fragments Polymerase chain reaction to rapidly isolate DNA fragments 0 PCR example, other slide Polymerase chain reaction to rapidly isolate DNA fragments Uses for PCR Genetic mapping Genotype detection Analyze traces of partially degraded DNA Evolutionary studies
Compare homologous sequences from related organisms Compare sequences from a variety of sources Studies of gene diversity Diagnosis of infectious diseases DNA Sequence Analysis All sequencing projects use same basic protocol. Sequence determined approximately 800 bases at a time. Maxim-Gilbert method Chemical cleavage of DNA at specific nucleotide types Enzymatic extension of DNA to defined terminating base Sanger method Sanger method most popular and efficient, particularly for automated methods Both techniques approximately 99.9% accurate General Principals of Sanger Sequencing Method General Principals of Sanger Sequencing Method 0 Sequencing example, other slide 0 Automated DNA sequencing 0 Automated DNA Output from an automated DNA sequencing reaction Each lane displays the sequence obtained from a separate DNA sample and primer. Automated DNA Sequencing Computer reads off the sequence complementary to the template strand Machine generates complementary strand Ambiguities are recorded as an "N" and can sometimes be resolved by a technician Sequencing long regions of DNA Primer walking
Sequence starting from both ends of cloned insert New primers derived from sequence obtained in previous round Sequencing long regions of DNA Shotgun sequencing Long DNA sequence chopped into many small fragments which are cloned individually Sequence of all small fragments determined Small fragments aligned by computer to generate one long continuous sequence Shotgun approach relies on redundancy. Rapid sequencing Must gather sequence information on 3-4 times the actual number of base pairs from the original clone for full coverage Sometimes must fill in gaps with primer walking Must have many automated sequencers Very fast if laboratory has enough equipment No redundancy required No alignment necessary Slower than shotgun sequencing because must make primers after each round of sequence Works well in laboratories without large number of automated sequencers Primer walking Understanding the genes for hemoglobin: a comprehensive example Recombinant DNA technology used to isolate the and globin gene loci Isolated RNA from red blood cell precursors Produced cDNA libraries Probed libraries for cDNA clones Sequenced individual cDNA clones to identify and globin coding sequences Used PCR to amplify sequences in many individuals with and without disease Probed genomic DNA libraries to identify genomic clones and surrounding regions of globin genes Used cDNA to probe Southern blots to determine number and location of coding sequences within genomic locus Sequenced entire chromosomal regions containing and globin genes The genes encoding hemoglobin occur in two clusters on two separate chromosomes The -globin cluster contains three functional genes that spans 28 kb on chromosome 16. The genes encoding hemoglobin occur in two clusters on two separate chromosomes The -globin cluster contains five functional genes and two pseudogenes spanning 50 kb on chromosome 11. The succession of genes in each cluster correlates with the sequence of expression during development globin cluster during the first five weeks of embryonic life Two chains during fetal and adult life globin cluster during first five weeks of embryonic life Two chains during fetal life and within a few months of birth Locus control region (LCR) turns genes on and off LCR associates with specialized DNA binding proteins at 5' end of each gene cluster, bending chromosome back on itself to turn genes on and off in order. Example where adult -globin genes are removed LCR cannot switch from activating fetal genes to activating adult genes. Fetal genes remain active in adult . A variety of mutations account for the diverse symptoms of globin-related diseases Two general classes of disorders Mutations alter amino acid sequence. Hemolytic anemias e.g., sickle cell anemia A-to-T substitution in sixth codon of -globin chain Mutations that reduce or eliminate production of one or two globin polypeptides thalassemia Hemolytic anemias Sickle Cell Anemia Thalassemias ...
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