Chapter 8—Manipulating Proteins, DNA, and RNA- Methods to analyze biological molecules and cells are constantly evolving and what we know in biology is often limited by the methods available - Biological molecules and cells can be examined within the context of the organism as well as outside the organism- Cells can be isolated, cultured, modified, and reintroduced into organisms- DNA can be separated by size, amplified, sequenced, modified, and reintroduced into cells or organisms; analogous approaches are used to analyze RNA - 'Proteins can be purified, tested for activity, and tested for interaction with othermolecules (including proteins). Their structure can be determined- Cells can retain some of their characteristics even when removed from tissue and grown in a dish- Embryonic stem cells can be used:- To generate all cell types of the bodyo Come from cells of inner cell mass (early embryo = blastocyst)cultured ES cells- For reproductive cloning or preserved for therapeutic cloningo Cells from adult tissue containing genome to be cloned mixes with nucleus from unfertilized egg cell fusioncell divisionearly embryoeither reproductive cloning or therapeutic cloning- Purifying proteins:- Centrifugation can be used to separate cells, organelles, and even large macromoleculeso Low-speed centrifugation (1000g, 10 min): produced pellet containing: whole cells, nuclei, cytoskeletono Medium-speed centrifugation (20,000g, 20 min): pellet contains mitochondria, lysosomes, peroxisomeso High-speed centrifugation (80,000g, 60 min): pellet contains microsomes, and small vesicleso Very high-speed (150,000g, 180 min): pellet contains ribosomes, viruses, and large macromoleculeso Separates components based on size (larger come out more at low speed)- DNA of a wide range of sizes can be separated using gel electrophoresiso Acrylamide gel (small pore)o Agarose gel (medium pore)o Pulsed-field agarose gel (periodic change in electric field)- The Polymerase Chain Reaction (PCR) can be used to amplify DNAo Oligonucleotides on 2 strands serve as primers for in vitro DNA synthesis and determine the segment of DNA to be amplifiedo Starts with double-stranded DNA – heat to separate – do over and over to producemore double-stranded DNA molecules- PCR of hypervariable microsatellites can be used in forensics for “DNA fingerprinting” Hypervariable microsatellites = a run of repeated nucleotides (variable number of tandem repeats- VNTR) Because of variability in these sequences at each locus – individuals usually inherit a different variant from their mother and from their father therefore two unrelated ppl do not contain the same pair of sequences- Dideoxy sequencing is a common method of determining the sequence of DNAo Relies on dideoxyribonucleoside triphosphates which lack the 3’ hydroxyl group prevents strand extension at 3’ end (versus deoxy-)o Purified DNA is synthesized in a mixture that contains single-stranded molecules of DNA to be sequences , the DNA polymerase, a short primer DNA, and 4 deocyribonucleoside triphosphates. If a dideoxy analog of one of these nucleotides is also present it can become incorporated into a growing DNA chain--< addition of next nucleotide is blocked = termination Produced DNA of different lengths terminated at different Aso To determine the complete sequence one strand is used as template for sequenceing 4 different chain-terminating dideocyribonucleoside triphosphates (ddATP, ddCTP, ddGTP, DDTTP) are used in 4 separate DNA synthesis reactions on the same single-stranded DNA template Products separated by gel electrophoresis to determine full sequenceo Dideoxy sequencing can be automated using chain terminating nucleotides with different fluorescent dyes- Analyzing and manipulating DNA:- DNA can be cut at specific sequences by many restriction nucleaseso To separate a gene from DNAo Enzymes cut DNA at recognition sequences with blunt or cohesive ends Compatible cohesive ends can be used to selectively anneal and ligate two different pieces of DNA Fragments with same cohesive ends cut by same enzyme can join by complementary base-pairing = recombinant DNAo Ligation by DNA ligase of two piece of DNA can generate recombinant DNA Circular DNA plasma DNA cut with restriction enzyme, DNA fragment tobe cloned is covalently linked by DNA ligase cloned plasmid- Recombinant plasmid DNA can be highly amplified by transfecting bacteria with the cloned plasmid- Then put bacteria in culture => many copies of purified recombinant plasmid isolated from lysed bacterial cells Large libraries of plasmids, each with a piece of human DNA, can be made: human genomic DNA library- DNA cleavage with restriction enzyme= million of genomic DNA fragments- DNA fragments inserted into plasmids= recombinant DNA molecules- Introduction of plasmids into bacteria = human genomic DNA library- mRNA can be converted into complementary DNA (cDNA) for analysis and storageo Reverse transcriptase produces DNA copies (cDNA) from mRNA- Genomic DNA libraries and cDNA libraries contain different sequenceso Genomic DNA library—both introns and nontranscribed DNA in clones, only part of coding sequence of a gene, genes represented equally regardless if one is more frequently transcribedo cDNA clones—introns removed by RNA splicing when mRNA made- DNA molecules can be labeled with radioactivity or chemical modificationso A. Purified DNA polymerase enzyme adds labeled nucleotides= population of DNA molecules that contain labeled examples of all sequences on both strands “body label” with radioactivity Can also be used to produce nonradioactice DNA molecules that carry a specific chemical marker that can be detected with an appropriate antibodyo B. Polynucleotide kinase labels only the 5’ end- when labeling followed by restriction enzyme cleavage the DNA molecules with only one 5’ end labeled can be obtained “end label” with radioactivity- Temperature can be manipulated to allow for strict or promiscuous interaction between two strands of DNAo To find an identical match the temp is kept below just below that at which a perfect DNA denatures in solvent so all imperfect helices are unstableo For related and identical sequences- temp is lower so imperfectly paired double helices form- Labeled probes can be used to identify where a sequence is present within a cell or organismo Mark their respected nucleotide sequences on
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