BSCI105- Chapter 20
BSCI105- Chapter 20 BSCI105
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This 8 page Class Notes was uploaded by Shira Clements on Monday April 18, 2016. The Class Notes belongs to BSCI105 at University of Maryland taught by Norma Allewell in Fall 2015. Since its upload, it has received 12 views. For similar materials see Principles of Biology I in Biology at University of Maryland.
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Date Created: 04/18/16
Shira Clements BSCI105 Chapter 20- Biotechnology Recombinant DNA- DNA molecules formed when segments of DNA from two different sources are combined into one, which helps to analyze genes and gene expression Biotechnology- manipulation of organisms to make useful products DNA Cloning Yields Multiple Copies of a Gene or Other DNA Segment - DNA is very long and carries many genes, but genes are small proportion of chromosomal DNA, so need methods to develop well defined segments, by DNA cloning o Usually uses bacteria, and its chromosome is large circular molecule of DNA, and have Plasmids- small circular DNA molecule that replicate separately from bacterial chromosome, with few genes. o To clone- first get isolated plasmid and then insert another source’s DNA into it o Which makes recombinant DNA molecule Then, plasmid is returned to bacterial cell, making recombinant bacteria Through divisions, clones are made and DNA is passed onto descendants o Useful- amplify gene and to produce a protein product Using Restriction Enzymes to Make Recombinant DNA - Gene cloning and genetic engineering rely on the enzymes that cut DNA molecules at specific locations, which are called endonucleases or restriction enzymes o Protect bacterial cell by cutting up foreign DNA from other organisms or phages o DNA is protected from cell’s own restriction enzymes by addition of methyl group o Usually palindromic when read in 5-3 direction o Have specific site to bind, which is called restriction site (short DNA sequence) o Cuts both DNA strands at precise points within restriction site o DNA of bacterial cell is protected from restriction enzymes by addition of methyl group to A or C within the sequences recognized by enzymes o Restriction fragments- when long DNA, restriction enzyme cuts it into fragments that happen to be the same o Most useful restriction enzyme cleave the sugar-phosphate backbones in the two DNA strands, resulting in one strand of double strand having sticky end- short extensions from DNA that form hydrogen bonds with complementary sticky ends on any other DNA molecules cut with same enzyme. They are joined together by DNA ligase (catalyze covalent bonds that close sugar phosphate backbones of DNA strand) Cloning a Eukaryotic Gene in a Bacterial Plasmid - Original plasmid is called a cloning vector- DNA molecule that can carry foreign DNA into a host cell and replicate there o Bacterial plasmids are used as cloning vectors because- 1. They are easily obtained 2. Can form recombinant plasmids by inserting foreign DNA 3. Multiply quickly o Method to clone Cells of Recombinant Plasmids- 1. Get engineered plasmid DNA and DNA from hummingbird cells, where the hummingbird’s DNA is the one that has DNA of interest 2. Plasmid and hummingbird’s DNA are cut by same restriction enzyme 3. Fragments mixed together, some join by simple base pairing of sticky ends, and ligase seals them (recombinant plasmid). Products include recombinant plasmids and many nonrecombiant plasmids 4. DNA mixture is added to bacteria that have mutation. In suitable conditions, cells take up foreign DNA by transformation 5. Plate the bacteria on nutrient. Only cells with plasmids will reproduce in these conditions because they are only ones with amp gene resisting ampicillin. And incubate until colonies grow. o Storing Cloned Genes- Starts with a mixture of fragments from entire genome, no single gene is targeted for cloning. Genomic library- complete set of plasmid-containing cell clones, each carrying copies of particular segment if initial genome. Like a book containing specific information Historically- bacteriophages have been used as cloning vectors for genomic libraries- fragments of foreign DNA can be spliced into a trimmed down versions of a phage genome, as into a plasmid by using restriction enzyme and DNA ligase Bacterial artificial chromosome (BAC)- another vector used in library construction- large plasmids, trimmed down so they contain just genes that are necessary to ensure replication Stored in wells o Cloning a Gene Genes is inserted into plasmid Plasmid is put into bacterial cell Host cell grown in culture to form a clone of cells containing the cloned gene of interest Basic research and applications Amplifying DNA in vitro- Polymerase Chain Reaction PCR - DNA cloning is still best method for preparing large amounts of genes, but this is quicker and more selective o Target segments can be quickly amplified o 3 steps in cycle- Denature- separate DNA strands by heat. Then cooled to allow… Anneal- H bonding of short DNA primers at the end of target sequence on both strands Extension- heat stable (key) DNA polymerase adds from primers at 3’ end and completes synthesis o After whole cycle, the number is doubled, so 2 , n= number of cycles o Product is often cloned into a plasmid o Cannot substitute for gene cloning when large amounts of genes are desired because there are occasional errors, which limits number of good copies Gel Electrophoresis- uses gel made of polysaccharide (agarose) and it separates the different genes by the size, the smallest going the farthest and the shortest falling not too far, showing bands DNA Sequencing- Dideoxy Method - Depends on random termination of DNA replication by dideoxy nucleotides - This produces a set of newly synthesized chains differing from one another by one nucleotide - The chains can be separated by gel electrophoresis and detected with fluorescent labels - Scanning the gels provides an image from which the DNA sequence can be read - Technique- o 1. fragments of DNA is denatured into single strands and incubated in test tube with ingredients for DNA synthesis- primer for 3’ end, DNA polymerase, and 4 deoxyribnucleotides, each tagged with specific fluorescent molecule o 2. Synthesis of each new strand starts at 3’ end of primer and continues until dideoxyribonucleotides is inserted, at random, instead of the normal deoxyribonucleotide. Eventually, a set of labeled strands of with lengths one nucleotide longer than the previous one will be generated with the color of the tag representing the last nucleotide in the sequence. o 3. Then put in gell electrophoresis which will get the right order of the strands, and we will then know what the sequence is because of the tags and everything Then, Next Gen Sequencing- uses nanobeads, PCR, robotics, and different detection method 1. Genomic DNA is fragments 2. PCR then copies each fragment, and attach to bead at 5’ end 3. Bead is then placed into a well with primer and DNA polymerase 4. Solution of each of the four nucleotides is added to all wells, and then the process is repeated after wells are washed 5. Primer is added to DNA, so DNA polymerase can begin synthesis o if the nucleotide is joined to growing strand in that solution (in A solution, so A would be the correct match), then there is a flash of light which is recorded o if the nucleotide in that solution is not complementary to the DNA strand, then nothing happens (no flash of light) Using DNA technology to Study Gene Expression and Gene Function - RT-PCR Analysis of Expression of Single Genes o Uses enzyme reverse transcriptase (makes cDNA from mRNA, an enzyme that can produce double stranded DNA from an RNA template) with PCR and gel electrophoresis. Because cDNA comes from mRNA, there are no introns, it is only exons- the final part. Can be used to produce eukaryotic proteins in bacteria o Used to compare gene expression between samples (different stages, tissues, or same cell in different conditions) o How? cDNA synthesis- mRNA is incubated with reverse transcriptase and other necessary components to make cDNA PCR amplification- cDNA is then entered into here using primers of specific gene that you want to amplify, and amount cDNA produced indicates the amount of mRNA present Gel electrophoresis- reveal amplified DNA products only in samples that contain mRNA transcribed from original gene Then results - In-situ Hybridization- o Another way to determine which tissues or cells are expressing certain genes and can track down the location of specific mRNA- detects specific mRNA o Uses DNA probe that is complementary to sequence in mRNA and is labelled, usually with a fluorescent dye, so that it can be visualized o We insert anticodons of specific DNA that we are looking for with fluorescent dye to tell you where in the organism it is- uses probes o Different probes, different dyes to distinguish locations o Homeotic genes are master regulatory genes that control placement and spatial organization of body parts in all eukaryotic organisms. In animals they can be recognized by a characteristic 180-nucleotide homeobox Studying Expression of Interacting Genes - DNA microarrays were the first successful technology - The microarray consists of thousands of genes - Two samples of labeled DNAs (eg red and green) are applied to the microarray - The color of the spot indicates which sample(s) bind to the gene at that spot Sample DNAs are cDNAs produced by viral reverse transcriptase from mRNAs extracted from A newer technology, RNA-seq, which uses reverse cells transcriptase and high throughput DNA sequencing is more versatile and potentially less subject to artifacts. Determining Gene Function Site directed mutagenesis ◦ Mutation is introduced into cloned genes, and then the mutated gene is then returned to the original, and knocks out the normal copies of that gene. Therefore, if there is a change, you can the function is then revealed ◦ (Modify codons in cloned gene; return the gene to the cell ◦ Function of cloned gene often examined in bacteria but can also be examined in original organism (except for humans!)) Next- Gen- RNAi ◦ Uses synthetic double stranded RNA that matches sequence of specific gene triggers breakdown of mRNA or blocks translation- prevent other genes from being expressed ◦ Can be carried out on a large scale, examining many genes Genome Wide Association Studies (GWAS) Goal: Define genetic basis of disease by analyzing genomes of large populations with specific phenotypic difference or disease Simplify by focusing on genetic markers o (differences in sequences that vary within population, as a result of different alleles (different sequences) for specific genes More specifically, focus on single nucleotide polymorphisms (SNPs): Single base pair change that is found in at least 1% of population ◦ Can be detected by PCR or microarray analysis without analyzing entire genome ◦ Most SNPs don’t cause disease but are useful markers for regions of chromosome Cloning Organisms lead to productions of Stem Cells Stem cells- unspecialized cells that can become a specialized cell and can reproduce itself indefinitely Organism Cloning- Taking an asexual producing cell and making it into a lot of identical cells Totipotent- any cell with potential to give rise to a specialize/differentiated cell - Dolly the sheep was the first animal cloned- they fused sheep eggs with dedifferentiated cells, which resulted in a dividing diploid cells, making o Took embryo from one sheep (haploid from mother and haploid from father), took out the nucleus (2 haploids) – took out the DNA o Dedifferentiated cells of a third part sheep of mammary cells by stopping the cell cycle (no longer specialized) o Put nucleus of dedifferentiated cell into the embryo of a donor sheep o Grow in culture o Implant into Uterus of another sheep o Clone (Dolly) is born Identical to the dedifferentiated cell from the third party sheep - Organismal cloning produces offspring from a single cell that are genetically identical to the source of the single cell - The most frequent current medical use of organismal cloning is in stem cell production - The first cells in embryos are stem cells - A stem cell is relatively undifferentiated, can reproduce indefinitely and can also differentiate into specialized cells (nerve, brain, muscle, etc) - Plants can be cloned relatively easily because their differentiated cells can de-differentiate into stem cells - The ability of differentiated animal cells to de-differentiate is limited. However, efforts are ongoing to find ways to induce de-differentiation - Cloned animals are generally produced by nuclear transplantation (nucleus from differentiated cell inserted into enucleated egg) - Cloned animals often have faulty gene regulation Why would you want to do organismal cloning? Produce stem cells that can be used to regenerate damaged tissue ( eg pancreas, brain, heart) Produce offspring that are genetically identical to the parents ◦ Satisfy the desire for immortality! ◦ Propagate plants and animals with desirable features (racehorses, livestock, agricultural plants, pets) Cloning animals Differentiated animal cells are generally not totipotent (can’t be used in organismal cloning) Totipotentency is gradually lost during development, as cells differentiate However, scientists are gradually learning how to turn differentiated cells back into induced pleuripotent stem cells (iPS cells) (can differentiate into some but not all cell types) Numerous false reports but also real progress Egg cell enucleated by UV light Donor nucleus transplanted from embryo (left), tadpole (right) Cell with embryonic nucleus differentiates into tadpole Cell with fully differentiated nucleus not able to proceed to tadpole stage Differentiated cells unable to de-differentiate and form other cell types Embryonic vs Adult Stem Cells Adults, as well as embryos contain stem cells However, adult stem cells have limited ability to turn into differentiated cells Nevertheless, adult stem cells have the ability to turn into several types of different cell types (eg brain stem cell-> nerves; bone marrow stem cells-> several types of blood cells or several types of tissue (bone, fat muscle, lining of blood vessels) New discoveries are constantly being made about how to increase the range of differentiated cells that can be produced
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