Download Chapter 20 Notes AP Biology I. Chapter 20.1: DNA - Pomp

Chapter 20 Notes AP Biology I.
Chapter 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment a. DNA Cloning and it Applications: i. Gene cloning: preparing well defined gene-­‐sized pieces of DNA in multiple identical copies 1. Allows a scientist to work with specific sequences of DNA ii. General features of DNA cloning: 1. The use of bacteria, E. coli and their plasmids a. Plasmids-­‐ small, circular DNA molecules that replicate separate from the bacterial chromosome b. To make a clone; the plasmid, it is first isolated and the foreign DNA is inserted. c. The resulting plasmid is a recombinant DNA molecule i. Contains DNA from two sources d. The plasmid is returned to the bacteria cell creating a recombinant bacterium which reproduces to form a clone of identical cells 2. Usefulness of cloned genes: a. Makes many copies of a particular gene b. Produces a protein product i. Can be used for research ii. Can be used to give a new organism new metabolic activities iii. Steps to DNA cloning: 1. Insert gene which codes for a desired protein into a plasmid to create a recombinant DNA plasmid 2. Plasmid is inserted back into the bacterium creating a recombinant bacterium 3. Host cell grows in a culture to from a clone of cells that contains the clone gene 4. Clone cells are used for research and various other applications a. Pest resistance in plants b. Oil eating bacteria c. Dissolving blood clots d. Human growth hormones to treat stunted growth b. Using restriction enzymes to Make Recombinant DNA i. Restriction enzymes: enzymes that cut DNA molecules at a limited number of specific locations 1. They cleave the sugar phosphate backbone ii. Discovered in the late 1960’s iii. Natural function is to protect the bacterial cell against intruding DNA from other organisms 1. Work by cutting up foreign DNA iv. Hundreds of restriction enzymes that are very specific 1. Restriction sites: short DNA sequences 2. Methylation to adenines or cytosines protects the bacterial cell from its own restriction enzymes (-­‐CH3) 3. Most restriction sites a symmetrical due to the 5’ to 3’ nature of the DNA molecule 4. Restriction sites contain between 4 to 8 nucleotide sequences-­‐ can occur many times in a DNA molecule so it fragments the DNA many times a. Makes many restriction fragments b. Each restriction enzyme produces the same restriction fragment v. Sticky ends: double stranded restriction fragments have at least one single stranded end 1. These short extensions can form hydrogen bonded base pairs with complementary sticky ends on any other DNA molecules cut with the same enzyme. 2. DNA ligase-­‐ catalyzes the formation of hydrogen bonds that close up the sugar-­‐phosphate backbones vi. Steps to using restriction enzymes: 1. Restriction enzyme cuts the sugar-­‐ phosphate backbones at a specific location on the DNA molecule 2. DNA fragment from another source is added a. Base pairing of sticky ends produces various combinations 3. DNA ligase seals the strands c. Cloning a Eukaryotic Gene in a Bacterial Plasmid: i. Cloning vector-­‐ a DNA molecule that can carry foreign DNA into a cell and replicate there 1. The original plasmid 2. Usually bacterial plasmids: a. Because they can be easily isolated b. Easily manipulated to form recombinant plasmids c. Easily reintroduced into bacterial cells d. Bacterial cells reproduce rapidly ii. Cloning a Eukaryotic Gene in a Bacterial Plasmid: 1. Isolate plasmid DNA from bacterial cells and DNA from human cells containing the gene of interest a. Example: ampR gene which makes E. coli cells resistant to the antibiotic amphicillin, and the lacZ gene which codes for β-­‐ galactosidase which hydrolizes sugar into glucose and galactose 2. Cut both DNA samples with the same restriction enzyme, one that makes a single cut within a specific gene and many cuts within the human DNA a. Example: the enzyme cuts the plasmid DNA at its single restriction site within the lacZ gene 3. Mix the cut plasmid and DNA fragments a. Some join by base pairing b. Add DNA ligase to seal them together c. Producing recombinant plasmids and many non-­‐
recombinant plasmids 4. Introduce the DNA into bacterial cells that have a mutation in their own gene a. Under suitable conditions, some cells will take up a recombinant plasmid or other DNA molecule by transformation b. Example: DNA is mixed with bacteria that have mutations in their own lacZ gene-­‐ making them unable to hydrolyze lactose 5. Plate the bacteria on agar containing the right nutrients and incubate until colonies grow. a. Only a cell that took up a the desired plasmid will form a colony 6. Recognizing cell clones carrying recombinant plasmids involves: a. Since the growth medium contains the antibiotic amphicillin, only those bacterial with the ampR will form colonies b. Only colonies with the non recombinant lacZ gene will turn blue because they will hydrolyze the X-­‐gel(lactose mimic) forming a blue product i. All other recombinant colonies with no functional β-­‐ galactosidase will be white 1. These contain the recombinant plasmids iii. Identifying Clones Carrying a Gene of Interest: 1. Nucleic acid hybridization: a. The DNA of the gene is detected by its ability to base-­‐pair with a complementary sequence on another nucleic acid molecule b. Nucleic acid probe: a short, single stranded nucleic acid that can be either RNA or DNA c. The complementary probe can be synthesized from a known sequence in the genome i. Each probe is labeled with a radioactive isotope or a fluorescent tag so we can track it. d. Steps to the nucleic acid probe hybridization: i. A special filter paper is pressed against the agar contain the desired colony containing the desired recombinant gene 1. The filter paper and agar is marked with X’s to establish the position of each colony in reference to the marks. ii. The filter is treated to break open the cells and denature their DNA 1. Resulting single stranded DNA to the filter 2. Radioactive probe molecules are incubated with the filter 3. The single stranded probe base pairs with any complementary DNA on the filter 4. Excess DNA is rinsed off iii. The filter is laid under photographic film, allowing radioactive areas to expose the film 1. Black spots on the film correspond to the locations on the filter of DNA that has hybridized to the probe iv. The film is developed and flipped over so the reference marks on the film and master plate are aligned so colonies carrying genes of interest are located. 2. Storing Cloned Genes in DNA Libraries: a. Genomic library: the complete set of plasmid clones carrying copies of a particular segment from the initial genome 3. Amplifying DNA in vitro: The Polymerase Chain Reaction (PCR) a. Polymerase Chain Reaction (PCR): a specific target segment within one or many DNA molecules can be quickly amplified in a test tube. i. Can make billions of copies of a target segment of DNA in a few hours 1. Quicker than searching a gene library for a clone and letting it replicate within host cells II.
ii. Three step cycle of PCR: 1. Cycle One: a. Denaturing: using heat to briefly separate DNA strands b. Annealing: Cool to allow primers to form hydrogen bonds with the ends of target sequence c. Extension: DNA polymerase adds nucleotides to the 3’ end of each primer. Must use a particular heat stable DNA polymerase isolated from bacteria found in hot springs d. Yields 2 molecules 2. Cycle Two: yields 4 molecules 3. Cycle Three: yields 8 molecules a. One fourth of the molecules match the target sequence b. Applications: i. 40,000 year old wolly mammoth ii. crime scene investigations iii. prenatal diagnosis of genetic disorders iv. detection of DNA of viral genes from cells infected with viruses Chapter 20.2: Restriction Fragment Analysis Detects DNA Differences that Affect Restriction Sites: a. Gel Electrophoresis and Southern Blotting: uses a gel as a molecular sieve to separate nucleic acids or proteins on the basis of size, electrical charge and other physical properties i. Nucleic acids and proteins are generally negatively charged ii. As they travel through the gel they are separated based on length iii. Restriction fragment analysis: when DNA fragments produced from restriction enzymes are sorted by gel electrophoresis iv. Allows for the preparation of pure samples of individual fragments v. Allows for the comparing of two DNA molecules-­‐ two alleles for a gene. b. Southern Blotting: i. Combination of gel electrophoresis and nucleic acid hybridization 1. Uses a radioactive probe of a single stranded DNA molecule that is complementary to the gene being analyzed ii. Steps to southern blotting of DNA fragments: used to detect specific nucleotide sequences within a DNA sample-­‐ analyzes specific restriction fragments produced from different samples of genomic DNA. 1. Preparation of restriction fragments a. DNA is mixed with restriction enzymes 2. Gel electrophoresis 3. Blotting: using capillary action, an alkaline solution is pulled upward through the gel and the DNA is transferred to a sheet of nitrocellulose paper (blot) and denaturing it in the process. a. The single DNA strands are stuck to the paper and positioned in bands corresponding to the gel 4. Hybridization with radioactive probe: the paper blot is exposed to a solution containing a radioactively labeled probe 5. Autoradiography: a sheet of photographic film is laid over the paper blot a. The radioactive probe exposes the film to form an image that corresponds to those bands containing DNA that base-­‐pairs with the probe. 6. Purpose: a. Can be used to identify heterozygous carriers of the sickle cell allele 7. Other blotting techniques: a. Northern blotting: detection of RNA sequences b. Western blotting: using proteins to detect protein bands i. Running serum proteins in a gel leaves smears instead of bands due to the large number of proteins with different sizes and charges ii. Western blot using antibodies specific for certain proteins c. Restriction Fragment Length Differences as Genetic Markers: i. Restriction Fragment Length Polymorphisms (RFLP’s) differences in the restriction sites on homologous chromosomes that result indifferent restriction fragment patterns 1. Scattered abundantly through out genomes 2. Can serve as a genetic marker for a particular location in the genome. III.
3. A given RFLP may occur in numerous variants in a population a. polymorphisms (Greek = many forms) 4. RFLP’s are detected and analyzed by the southern blotting technique. d. DNA sequencing: i. Sanger method: 1. Developed in 1977 by Fredrick Sanger a. Used for 25 years b. Replaced by the Next Gen sequencing methods c. Still used for small scale projects 2. Requirements: a. Single stranded DNA template b. DNA primer c. DNA polymerase d. dNTP (deoxynucleosidetriphosphates) i. dATP ii. dGTP iii. dCTP iv. dTTP e. ddNTP: dideoxyNTP’s-­‐ terminate DNA strand elongation i. lack a 3’ –OH group required for the phosphodiester bond ii. causes DNA polymerase to stop adding nucleotides 3. Steps to the Sanger Sequencing Method: a. Unknown DNA is separated into two strands b. Strands are copied using chemically altered bases i. This causes the copying process to stop when one particular base is encountered ii. The process is carried out for all four bases c. Fragments are put together like a gigsaw puzzle Chapter 20.3: Entire Genomes can be mapped at the DNA level: a. Human Genome Project: i. The sequencing of the human genome ii. Started 1990 finished in 2003 iii. Researchers have also been working on the genome of other species 1. Yeast: Sacchromyces cerevisiae 2. Nematodes: Caenorhabditis elegans 3. Drosophila melanogaster: fruit fly 4. Mus musculus: mouse b. Genetic (Linkage) Mapping: Relative Ordering of Markers i. FISH: fluorescence in situ hybridization 1. Fluorescently labeled probes are allowed to hybridize to an immobilized array of whole chromosomes a. Chromosomes tagged to reveal a specific chromosome ii. Cytogenic maps-­‐ based on the information provided by FISH provide the basis for more detailed mapping 1. Chromosome banding pattern and location of specific genes by FISH iii. Linkage map: map of several thousand genetic markers space through out the chromosome 1. Ordering of genetic markers such as RFLPs 2. Simple sequence DNA 3. Other polymorphisms-­‐ about 200 per chromosome c. Physical Mapping: Ordering DNA Fragments i. The distances between markers are expressed in some physical measure, usually the number of base pairs along the DNA 1. Mapping the whole genome a. Physical map is made by cutting the DNA of each chromosome into restriction fragments b. Determining the original order of the fragments, overlapping them c. Then using probes or automated nucleotide sequencing of the ends to find the overlaps d. Materials for mapping are prepared by cloning e. Cloning vectors: i. YAC-­‐ yeast artificial chromosome-­‐ can carry inserted fragments of a million base pairs long ii. BAC-­‐ bacterial artificial chromosome-­‐ can carry and insert 100,000 to 500,000 base pair fragments f. The ordering is followed by ordering of smaller fragments cloned in phage and plasmid vectors d. DNA sequencing: i. Ultimate goal is determining the direct order of nucleotide sequence in a genome. ii. Dideoxyribonucleotide chain termination method: iii. Technique: 1. Each strand starts with a primer and ends with a dideoxyribonucleotide(terminates a growing DNA strand because it lacks a 3’ –OH group) 2. Each ddNTP is tagged with a fluorescent marker, the identity of the ending nucleotides of the new strands and ultimately the entire original sequence can be determined. iv. Steps: 1. DNA to be sequenced is denatured into single strands a. Incubated in a test tube with the necessary ingredients i. Primer ii. dNTP’s(four of them: dATP, dGTP, dCTP and dTTP) iii. DNA polymerase iv. ddNTP’s(four of them: ddATP, ddGTP, ddCTP, ddTTP) 1. each tagged with a fluorescent marker 2. Synthesis starts at the 3’ end of the primer and continues until the ddNTP is inserted at random a. This prevents further elongation of the strand b. Each ddNTP is labeled with a colored fluorescent tag 3. Labeled strands are separated by passage through a polyacrylamide gel in a capillary tube. a. The shorter strands move through at a faster rate 4. A fluorescent detector senses the color which is picked up by a detector. e. The human genome project was moved forward by the development of technology for faster sequencing and more sophisticated computer software for analyzing and assembling the partial sequences.