Biology Chapter 20 - Biotechnology
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| 1095014531 | DNA Cloning | Procedure in which a fragment of DNA is inserted into a vector and then transferred to another cell, where it then replicates. (Multiple copies of a single gene = gene cloning) |  |
| 1095014532 | Restriction Enzymes (make Recomb. DNA) | Cut DNA at specific nucleotide sequences. Instead of cutting the molecules straight across, they leave "sticky ends". Any DNA cleaved by the such enzyme can be joined readily to another DNA molecule cleaved by the same enzyme. ( ** DNA ligase is used for permanent union) | |
| 1095256933 | Plasmids | An circular !self-replicating! non-chromosomal DNA molecule found in many bacteria, capable of transfer between bacterial cells of the same species, and occasionally of different species. Antibiotic resistance genes are frequently located on plasmids. Plasmids are particularly important as vectors for genetic engineering | |
| 1095256934 | Bacterial Artificial Plasmid | A plasmid in bacteria that can hold large segments of DNA (>100 kb) and contains genes for partitioning the plasmid to daughter cells, antibiotic resistance genes, and an origin of replication | |
| 1095094411 | Gene Cloning in Bact. Plasmid | Cloning vector - the DNA molecule (plasmid) that can carry foreign DNA into a host cell and replicate there. 1. Isolate genomic DNA of interest and obtain bacterial plasmid for vector. Plasmid can contain genes like ampR for antibiotic resistance and lacZ which can hydrolyze lactose. 2. Plasmid and DNA of interest are cut with same restriction enzyme. 3. Fragments are mixed together and DNA ligase is added. 4. DNA mixture is added to bacteria. 5. Selection is done by phenotypes such as amp resistance or lactose consumption. | |
| 1095266762 | Transfection | electroporation: A technique to introduce recombinant DNA into cells by applying a brief electrical pulse to a solution containing cells. The electricity creates temporary holes in the cells' plasma membranes, through which DNA can enter. Viruses/gene guns/thin needles: Can also be used to transform cells via injection of DNA Chemical methods can also interact with the cell membrane to make it permeable to DNA insertion. | |
| 1095094412 | cDNA from mRNA procedure | Fully processed mRNA is extracted from cells where the gene is expressed. Reverse transcriptase (obtained from retroviruses) is used to make a single stranded DNA reverse transcript of each mRNA molecule. DNA polymerase is used to create the complementary strand (poly A tail allows use of short strand of dT's to be used as primer). = complementary DNA - cDNA. | |
| 1095094413 | cDNA Library (vs Genomic library) | cDNA libraries won't include introns/exons or other regulatory elements. But a cDNA library is useful for studying sets of genes expressed in particular cell types. Also making cDNA at different time points in a cell allows tracking of patterns of gene expression during development. | |
| 1095094414 | Nucleic Acid Hybridization | A technique in which a single-stranded nucleic acid probe is made that is complementary to, and binds to, a target sequence, either DNA or RNA. The resulting double-stranded molecule is a hybrid. The probe can be labeled with a radioactive isotope, a fluorescent tag, or another molecule for tracking. | |
| 1095094415 | Polymerase Chain Reaction (PCR) | DNA Amplification in Vitro - PCR Double stranded DNA containing target sequence + heat resistant DNA polymerase, all four nucleotides, two DNA primers for target sequence (one for each end). => thermocycler: One cycle yields 2 molecules: (2^n where n is cycles) 1. Denature: Heat denatures DNA (seperate strands) 2. Anneal: Cool allowing primers to form bonds with DNA 3. Extension: DNA polymerase adds nucleotides to 3' end of primers extending sequence. | |
| 1095167156 | Gel Electrophoresis | The separation of nucleic acids or proteins, on the basis of their size and electrical charge, by measuring their rate of movement through an electrical field in a gel (such as agar). DNA moves towards positive pole due to their negative charge (phosphate groups). | |
| 1095167157 | Southern Blotting | Gel electrophoresis + nucleic acid hybridization = detect bands that include only parts of gene of interest: 1. Prepare fragments: Restriction enzymes digest samples 2. Gel electrophoresis: pattern of bands created 3. DNA transfer (blotting): DNA is transfered to a membrane creating a blot with a pattern of DNA bands just like those of the gel 4. Hybridization: The blot is exposed to a solution containing labeled probe. 5. Detection: sheet of film is used to detect hybrids. | |
| 1095228510 | Western Blotting | technique that uses antibodies to detect the presence of specific proteins separated by electrophoresis | |
| 1095228511 | Gels vs Gels | Agarose gels: DNA/RNA electrophoresis, uses size (compare to size ladder), visualize w/ ethidium bromide. Poly-acrylamide gels: (SDS)PAGE, proteins, uses size/blobbiness (compare to size ladder), visualize with Commaise blue or antibody -> Western blot! | |
| 1095208651 | DNA Sequencing | is a technique for determining the precise order of the nucleotides in a DNA fragment. | |
| 1095208653 | Dideoxy Chain Termination (Sanger Method) | Used to determine DNA sequence by inducing replication with altered bases that cause fragments of various sizes to be produced. Creates a timed result printout that is read from bottom to top (bottom being the last nucleotide of shortest labeled strand). See image. |  |
| 1095208657 | Northern blotting | Study expression of single genes: A technique that enables specific nucleotide sequences to be detected in a sample of mRNA. It involves gel electrophoresis of RNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe. | |
| 1095208659 | Reverse transcriptase PCR | Quicker and more sensitive than Northern blotting (requires less mRNA). Reverse transcriptase yields cDNA which is used as a template for PCR amplification using primers. Then, product is run on a gel and copies of the amplified region will be observed as bands only in samples that contain the mRNA of the gene interest (to measure gene expression). | |
| 1095208662 | in situ hybridization | a method for detecting particular mRNA transcripts in tissue sections by providing a nucleotide probe that is complementary to, and will therefore hybridize with, the transcript of interest. |  |
| 1095256935 | DNA Microarray Assay | A method to detect and measure the expression of thousands of genes at one time. Tiny amounts of a large number of single-stranded DNA fragments representing different genes are fixed to a glass slide. These fragments, ideally representing all the genes of an organism, are tested for hybridization with various samples of cDNA molecules. 1. Isolate mRNA, make cDNA via RT using labeled nucleotides. 2. Apply cDNA to microarray w/ different gene in each spot. cDNA hybridizes with complementary DNA (probes) on the microarray. 3. Rinse off excess cDNA. Scan for fluorescence. Positive fluorescence = gene expressed in the sample. | |
| 1095289761 | Organismal Cloning | the same as reproductive cloning - a method to create a clone of an entire multicellular organism; this is done by taking a donor cell and fusing it with an egg cell whose nucleus has been removed; the fused cell begins dividing normally; this embryo is then placed in a foster mother's uterus and the embryo develops normally | |
| 1095289762 | Cell Potency | Stem cells - undifferentiated cells that continue to divide. They are pluripotent Pluripotency - ability to differentiate into MANY different cell types. Totipotency - ability to differentiate into ANY cell type. | |
| 1095289763 | Nuclear Transplantation | a technique in which the nucleus of one cell is placed into another cell that already has a nucleus or in which the nucleus has been previously destroyed (enucleated). Can be used for reproductive cloning of a mammal (e.g. Dolly). | |
| 1095329693 | Cell Reprogramming | Increase in potency, dedifferentiation. Can be induced by nuclear transfer, cell fusion, genetic manipulation. Differential success rates in use of embryonic cells versus adult mammalian cells in reproductive cloning of a mammal indicates nuclear changes to cells over time with development (epigenetic changes: acetylation of histones, methylation of DNA etc. cause low success rates for normal development. | |
| 1095329694 | Induced Pluripotent Stem Cells (iPS Cells) | Any cell, even a highly differentiated cell in the adult body, that has been genetically reprogrammed to mimic the pluripotent behavior of embryonic stem cells. Done via delivery of certain transcription factors. -Can differentiate into all three germ layers (ecto meso end -derms). -Telomerase activity -Stem cell markers Still impossible to completely reverse all epigenetic changes. | |
| 1095329695 | Transgenics | the study and practice of genetic modification by inserting genes from one species into the genetic material of another species. This can produce a new "designed" species. Can be used for protein production by "pharm" animals. | |
| 1095329696 | Human gene therapy | an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery. |
