3rd exam notes 2 - Using restriction enzymes to make...

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Using restriction enzymes to make recombinant DNA Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites, yielding restriction fragments Many restriction enzymes cut DNA in a staggered way, producing fragments with “stick ends” A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell A cDNA library represents only part of the genome- only the subset of genes transcribed into mRNA in the original cells cDNA avoids introns… Amplifying DNA in vitro: The polymerase chain reaction (PCR) The PCR can produce many copies of a specific target segment of DNA A three step cycle- heating, cooling, and replication, doubles the amount of DNA every cycle DNA technology allows us to study the sequence, expression, and function of a gene Several techniques are used to analyze DNA Gel electrophoresis Rapidly analyzes and comparing genomes Uses gel as a molecular sieve to separate nucleic acids or proteins by size Restriction fragment length polymorphism RFLP analysis, DNA fragments produced by restriction enzyme digestion is sorted by gel electro… Restriction fragment analysis is useful for comparing DNA molecules, such as two alleles for a gene
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Single nucleotide polymorphisms (SNPs) are useful genetic markers These are single base paris that vary in a population When a restriction enzyme Diagnosis of diseases Genetic disorders can be tested for using genetic markers that are linked to the disease-causing allele Huntington’s disease is an example. We now have a test for the mutant allele that relies on a nearby SNP. DNA sequencing Relatively short DNA fragments can be sequenced by the dideoxy chain termination method (after Sanger) Dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths Cloning organisms may lead to production of stem cells for research and other applications Organismal cloning produces one or more organisms that are identical to the parent cell Reproductive cloning of mammals In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult shep by nuclear transplantation from a differentiated mammary cell Dolly’s premature death in 2003, caused because they used an aging cell to produce the new organism.. so dolly was born an adult sheep genetically, and got aging problems, such as arthritis, very early Since 1997, reproductive cloning has been done in many mammals Problems with animal cloning
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In most studies, only a small percentage of cloned embryos have developed normally to birth Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development Stem cells of animals
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