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Manipulating nucleic acids


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Manipulating nucleic acids

  1. 1. Biochemical and Molecular Methods Hybridization techniques allow identification of specific macromolecules, e.g. proteins, and DNA or RNA sequences based on ability of NA to bind specifically to labeled, known, single- stranded NA sequences. Separation methods employed range from enzyme digestion, chromatography, electrophoresis or centrifugation. Using restriction endonucleases which cleave DNA at specific sequences, recombinant DNA and other facets of biotechnology are benefited by these methods. Wait until next semester for this…
  2. 2. Nucleic Acid Hybridization. (A) If the DNA helix is separated into 2 strands, the strands should reanneal, given the appropriate ionic conditions andtime. (B) If DNA is separated into its 2 strands, RNA should be able to bind to the genes that encode it. If present in sufficiently large amounts compared with the DNA, the RNA will replace one of the DNA strands in this region.
  3. 3. Combined with reporter molecules, hybridization enablescytogeneticists to create probes to detect, quantify, visualize DNA/RNA location, and monitor their activity in a given cell/ tissue of interest.
  4. 4. Construction of a human genomic DNA library. A genomic library is usually stored as a set of bacteria, each bacterium carrying a different fragment of human DNA. The entire collection ofclones derived from one mRNA preparation constitutes a cDNA library. Because the cells of different tissues produce distinct setsof mRNA molecules, a distinct cDNA library is obtained for each type of cell used to prepare the library. http://highered.mcgraw-
  5. 5. The synthesis of cDNA.Total mRNA is extracted from a particular tissue. The enzymereverse transcriptase produces DNA copies (cDNA) of the mRNA molecules. A shortcomplement to the poly-A tail at the 3 end of the mRNA acts as a primer for the reverse transcriptase, which then copies the RNA into a complementary DNA chain, thereby forming a DNA/RNA hybrid helix. Treating the DNA/RNA hybrid withRNaseH creates nicks and gaps in the RNA. DNA polymeraseused to synthesize the 2nd DNA strand synthesize the bound RNA molecule, resulting to sequences at the 5„ end to be absent from cDNA libraries.
  6. 6. Gene A is infrequentlyThe differences between cDNA clones transcribed compared toand genomic DNA clones derived from gene B. In the genomic the same region of DNA. DNA library, both the introns (green) and the nontranscribed DNA (pink) are included in the clones. Most clones contain only part of the coding sequence of a gene (red). In the cDNA clones, the intron sequences (yellow) have been removed by RNA splicing during the formation of the mRNA (blue). Because gene B is transcribed more frequently than gene A in the cells from which the cDNA library was made, it is represented much more frequently than A in the cDNA library. In contrast, A and B are in principle represented equally in the genomic DNA library.
  7. 7. Preparation of a bacteriophage λ cDNA library. A mixture of mRNAs is isolated and used to produce cDNAs corresponding to all the cellular mRNAs (1–3). These single-stranded cDNAs (light green) are then converted into double- stranded cDNAs, which are treated with EcoRI methylase to preventsubsequent digestion by EcoRI (4 –6 ). The protected double-stranded cDNAs are ligated to a synthetic double- stranded EcoRI-site linker at both ends and then cleaved with the corresponding restriction enzyme, yielding cDNAs with sticky ends (red letters); these areincorporated into λ phage cloning vectors, and the resulting recombinant λ virions are plated on a lawn of E. coli cells (7–9 ) http://highered.mcgraw-
  8. 8. Phage cDNA libraries can be screened with aradiolabeled probe to identify a clone of interest. The appearance of a spot on the autoradiogram indicates thepresence of a recombinant clonecontaining DNA complementary tothe probe. The position of the spot on the autoradiogram is themirror image of the position on the original petri dish of that particular clone. Aligning the autoradiogram with the original petri dish will locate the corresponding clone from which infectious phage particles can be recovered and replated at lowdensity, resulting in well-separated plaques. Pure isolates eventually are obtained by repeating the hybridization assay.
  9. 9. Use of PCR to obtain a (A) PCR primers that flank the stretch of DNA to be cloned aregenomic or cDNA clone. added to purified chromosomal DNA, and many PCR cycles of are completed. Since only the DNA between the primers is amplified, PCR provides a way to obtain a short stretch of chromosomal DNA selectively in a virtually pure form. (B) To use PCR to obtain a cDNA clone of a gene, mRNA is first purified from cells. The first primer is then added to the population of mRNAs, and reverse transcriptase is used to make a complementary DNA strand. The 2nd primer is added, and the single-stranded cDNA molecule is amplified through many PCR cycles. For both types of cloning, the nucleotide sequence of at least part of the region to be cloned must be known beforehand.
  10. 10. Cloning genomic DNA by the PCR technique. Each cycle of the reaction begins with a brief heat treatment to separate the two strands (aka,heat cycler). Hybridization to complementary sequences in the two DNAstrands take place to produce 4 dsDNA molecules, and the 5-min. cycleis repeated 20-30x to produce amplified DNA copies. Trace amounts of RNA can be analyzed in the same way by first transcribing them into DNA with reverse transcriptase.
  11. 11. ......geneticsvideos http://highered.mcgraw- pcr.exe icro15.swf. A PCR reaction using 2 primers that bracket a particular microsatellite, or VNTR sequence, produces a different pair of DNA bands from each individual. Each band represents VNTR sequences inherited from the mother and father. Although some individuals have several bands in common, the overall pattern is quite distinctive for each.
  12. 12. The PCR cloning Bands obtained from a set technique has of PCR reactions which largely replaced amplifies different VNTRSouthern blotting sequences, can serve asfor the diagnosis a "fingerprint" to identify of genetic each individual. The diseases and for starting material for the the detection of PCR reaction can be alow levels of viral single hair or blood left at infection. the crime scene.
  13. 13. Methods for mRNA Isolate populations of mRNA that characterize certain cell types and are absent in all others Determine the temporal and spatial locations of RNA expression1.Northern blot- extract total mRNA from the specimen and separate it by electrophoresis. Bands produced are complementary mRNA to the probe, and intensity of bands are proportional to amount of specific mRNA.
  14. 14. Northern blotting.
  15. 15. 2.Ribonuclease protection 32Plabeled probe is hybridized insolution with total RNA from specimenHybrids are digested with ribonucleases (double-stranded ones will be protected from digestion) The mixture is separated on a sequencing gel Visualized radioactivity reveals a range of intensity proportional to the content of specific RNA in the sample.
  16. 16. 3.Reverse-transcription polymerase chain reaction (RT-PCR) Total RNA from sample is reverse-transcribed into cDNA using reverse transcriptase. Two oligonucleotides are added, chosen to correspond to sequences in the target cDNA. The sequence between the primers is amplified byrepeated cycles of synthesis, melting and hybridization. The reaction mixture is run on a gel and the DNA bands are visualized in the usual way. The intensity of bands bears some relation to the initial amount of mRNA in the sample.
  17. 17. Methods for DNA1.In situ hybridization- designed to reveal the spatial domains of gene expression in a specimen. An antisense probe is synthesized in vitro complementary to the mRNA to be detected. This is hybridized to the specimen and then visualized. Probes usually include extra chemical groups recognizable by a commercially available antibody for detection (e.g. digoxigenein or DIG, a plant sterol) Radioactive probes are used in radioactive in situ hybridization (RISH), fluorescent dyes are used in FISH.
  18. 18. Fluorescence In SituHybridization (FISH) begins with a DNA probe and a targetsequence. The DNA probe is labeled by indirect labeling (left) and directlabeling (right). The labeled probe and the target DNA are denatured to yield single strandedDNA. They are then combined, whichallows the annealing of complementary DNA sequences.
  19. 19. In situ hybridization performed on a whole chick embryo that have been fixed without being sectioned. The probe used recognizes the mRNA encoding Pax6 in the chick embryo. This probe is labeled not with a radioactive isotope, but with amodified UTP. To create this probe, a region of the cloned Pax6 gene was transcribed into mRNA, containing UTP conjugated with digoxigenin. This does not interfere with the coding properties ofthe resulting mRNA, but does make it recognizably different from any other RNA in the cell.
  20. 20. 2.Southern blot- to evaluate DNA extracts from tissue samples. This is a type of nucleic acid hybridization test in which single-stranded DNA from two sources interact. Strands with similar nucleic acid sequences will anneal by base pairing (A with T, and G with C) to form double-stranded molecules. One of the single-stranded DNA molecules is a unique portion of the gene of interest, and is radioactively labeled so it can be detected on photographic film (the probe).
  21. 21. Southern blotting. (1) DNA is treated with restriction enzymes, and the resulting restriction fragments of DNA are placed in a gel. (2) After the fragments of DNA are separated on the gel by electrophoresis, the DNA is denatured into single strands. (3) The gel is then placed on a support on top of a filter paper saturated with high-ionic-strength buffer. Nitrocellulose paper or a nylon filter is placed on the gel, and towels are placed atop the filter. The transfer buffer makes its way through the gel, nitrocellulose paper, and towels by capillary action, taking the DNA with it. The single-stranded DNA isstopped by the nitrocellulose paper. (4) Blot is incubated with radioactive or fluorescent probes in sealed bag. (5) The positions of the DNA in the paper directly reflect the positions of the DNA fragments in the gel.
  22. 22. *Blue dress of White House employee Monica Wilsonstained with semen of former US President Bill Clinton!
  23. 23. Southern blots (zoo blots) of various organisms. DNA using aradioactive probe from the Antennapedia gene of Drosophila melanogaster.Autoradiography shows that Drosophila genes contain several portions that are like Antennapedia genes in structure and that many organisms containseveral genes that will hybridize this radioactive gene fragment, suggestingthat Antennapedia-like genes exist in these organisms. The numbers beside the blots indicate size of bands, in kilobases.
  24. 24. 3.MICROARRAYS provide a means to measure & monitor the expression of thousands of genes at once http://highered.mcgraw-
  25. 25. DNA microarray analysis can reveal differences in gene expression in yeast cells under different experimental conditions. cDNA prepared from mRNA isolated from wild-typeSaccharomyces cells grown on glucose or ethanol is labeledwith different fluorescent dyes. If a spot is , expression of that gene is the same in cells grown either on glucose or ethanol. If a spot is , expression of that gene is greater in cells grown in glucose. If a spot is , expression of that gene is greater in cells grown in ethanol.
  26. 26. 3. GENE TARGETING (KNOCK-OUT) EXPERIMENTS- wild type alleles are replaced with mutant ones. There are 2 types of mutations used in these experiments: a. Loss of function mutation- protein product of the mutant gene is less active than the wild type b. Gain of function mutation- mutant gene interferes with the function of the wild-type form (mutant can cause receptor activation in the absence of a ligand-receptor complex, or a mutant transcription factor may be active all the time and not respond to regulation)
  27. 27. METHODS OF RECOMBINANT DNA TECHNOLOGY Knowledge of the molecular biology of cellsmakes it possible to experimentally move from gene to protein and from protein to gene!
  28. 28. 1. Bacterial plasmids are small, circular, self- replicating, extra-chromosomal DNA pieces that can be altered in vitro by inserting or deleting specific sequences, using restriction endonucleases. Because they can be used to create clones of genes, plasmids are called CLONING VECTORS. http://highered.mcgraw- ......geneticsvideosrestriction.exe
  29. 29. Transfection- cells are incubated in solution that makes them “drink” in cloned DNAusually mixedwith antibiotic resistance genes
  30. 30. Electropo lation- high- voltage pulse“pushes” the clonedDNA into cell
  31. 31.  Microinjection- cloned gene in solution is injected into the cell nucleus. The DNA is injected into the fertilized ovum before the male and female pronuclei have fused, increasing the probability that all of the cells of the organism will harbor the gene. A “gene gun” is also used which fires plastic bullets filled with DNA-coated metallic pellets. Some may penetrate the nuclei of cells, where the introduced DNA integrates into the DNA of the recipient‟s genome.
  32. 32.  Transposable element or retroviral vector- cloned DNA is inserted into mobile regions of DNA that has the ability to integrate themselves into the genome of an organism. Known as jumping genes since they can move about on the chromosome or among chromosomes.
  33. 33. Transgenic mice are produced by randomintegration of a foreign gene into the mouse germ line. Foreign DNA injected intoone of the two pronuclei (the male and female haploid nuclei contributed by the parents) has a good chanceof being randomly integratedinto the chromosomes of the diploid zygote. Because a transgene is integrated intothe recipient genome by non- homologous recombination, it does not disrupt endogenous genes.
  34. 34.  After birth, tissue samples of the young are assessed for the presence of the desired gene. DNA from germ line cells is given special attention. If the novel gene is present in these cells, the animal and its stem cells can be used as a founder for breeding. Such animal models allow researchers to test therapeutic compounds and study the molecular basis of given diseases. Mouse disease models now exist for cystic fibrosis, beta- thalassemia, atherosclerosis, retinoblastoma, and Duchenne muscular dystrophy. Somatic cells may also be grown in cell culture and genetically modified by fusion with the enucleated egg. With donor DNA for cloning derived from cultured recombinant cells, it becomes possible to carry out specific genetic modifications and introduce the modified genes into animals and plants.
  35. 35. Isolating embryonic stem (ES) cells and incorporating theminto recipient embryos resulting into cells of different genetic constitution appearing in the same organism (chimera). ES cells treated as transgenics have been useful in determining how genes are regulated during development of mice.
  36. 36.  To analyze the role of BMP7 in development, a bacterial gene for neomycin resistance is inserted into BMP7, destroying its ability to function. The mutant BMP7 genes are inserted into neomycin sensitive ES cells, heterozygous ES cells are then microinjected into mouse blastocysts, resulting to chimeras which are then mated to wild-type mice. Heterozygous mice are inbred, producing mutant mice which lacked eyes and kidneys.
  37. 37. In the absence of the BMP7 protein, cells thatform the eyes & kidneys stop dividing and die. Gene targeting makes transgenic mice that aremissing specific genes. In this way gene targeting can be used to analyze the roles of particular genes during mammalian development. .......geneticsvideosgene targeting.exe
  38. 38. Today, transgenics arebecoming such a common commodity. GMO technology comes up with new wonders every now and then, it is notsurprising to have one of these in the future!
  39. 39. 4. ANTI- SENSE RNA- interrupts translation of mRNA to protein by introducing single strands of RNA targeted to bind with the mRNA Generated by cloning DNA into vectors with promoters at both ends of the inserted gene. When incubated with RNA polymerase and NTPs, the promoter will transcribe the message in the wrong direction. Transgenic tomatoes have been constructed that carry in their genome an artificial gene that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.
  40. 40. The double-stranded RNA molecules arerecognized by an RNaseand degraded into short fragments.This antisense RNA willbind to a normal cellular message. mRNA produced by this gene will also be degraded. RNA-dependent RNApolymerase can amplifythese fragments, which can be transmitted to progeny cells. Results are similar toknock-out experimentswhere the expression of a cellular gene isexperimentally shut off.
  41. 41.