Chapter 20: Recombinant DNA Technology Summary (Page 1)- Recombinant DNA refers to putting together DNA molecules that doesn’t normally come together in natureo Method used to copy or clone DNA is called recombinant DNA technologyo Restriction enzymes: like a scissor, protective mechanism that restricts what stuff comes in thecell, cut DNA at specific recognition sequenceso Vector: a mechanism that replicates DNA, DNA molecules that accept DNA fragments and replicated inserted DNA fragments when vectors are placed into host cells o Clone: specific piece of DNA, not the entire animalo MicroRNA in cancer turns down the expression of oncogeneso Restriction site: a specific nucleotide where a restriction enzyme recognizes and binds to DNAo Palindrome: the nucleotide sequence that reads the same on both strand of the DNA when read in the 5’-3’ directiono Cleaved DNA structures by endo and exonuclease can be: Cohesive ends: sticky ends Blunt ends: clear cuto Anneal means to stick together by hydrogen bondingo DNA ligase seals the DNA fragments back together after cleavage (seal phosphodiester bond)o Selectable marker genes are used to distinguish host cells that have taken up vectors from host cells that have noto Genetically modified Bacterial Plasmids were the first vectors and replicated multiple copies Plasmid cloning vectors were derived from naturally occurring plasmids Two techniques for bacterial transformation (when plasmids are introduced to bacteria)- Treating calls with calcium ions and using a brief heat shock to pulse DNA into cells- Electroporation uses a brief, but high intensity, pulse of electricity to move DNA into bacterial cellso Multiple cloning site: allow scientists to clone a range of different fragments generated by many commonly used restriction enzymes.o Cells carrying recombinant plasmids can be selected my plating on medium containing antibiotics and color indicators such as X-gal X-gal is a substrate for lacZ turning it blueo Ampicillin is a common antibiotic used in these methodso Lambda phage: a virsus that infect E. Coli, has been completely mapped and sequencedo Bacterial artificial chromosomes: essentially very large but low copy number plasmids that can accept DNA insertso Yeast artificial chromosomes: can clone NA inserts much larger than BAC’so Expression Vectors are designed to ensure mRNA expression of cloned gene with the purpose of producing many copies of the gene’s encoded protein in a host cello Ti-plasmid (tumor inducing) has a part known as T-DNA where segment control tumor formation and the synthesis of compounds required for growth of the infect bacteriao Agrobacterium are transformed into restriction sites inTI plasmids that can be used to insert foreign DNA and recombinant vectors- DNA libraries contain many overlapping fragments of the genome, in turn span the entire genomeo Complementary DNA libraries offer certain advantages over genomic libraries because it contains DNA copies make of mRNA molecules of only expressed genes. Reverse transcriptase uses the mRNA as a template to synthesize a complementary DNA strand- Polymerase Chain Reaction revolutionized recombinant DNA methodologyChapter 20: Recombinant DNA Technology Summary (Page 2)o A rapid method of DNA cloning that extends the power of recombinant DNA research and in many cases eliminate the need to use host cells for cloning Typically follows three steps: denaturation, hybridiazation/annealing- Quantitative real-time RCR: quantify reactions as they occur in “real time,” made it possible to determind the amount of PR product made during the experiment- Reverse Transcription PCR: RNA is isolated from the cells or tissues to be studied and reverse transcriptase is used to generate double strand cDNA molecules- Restriction map: establishes the number of, order of, and distances between restriction-enzyme cleavage sites along a cloned segment of DNA- Electrophoresis uses an agarose gel to separate DNA using an electric charge- Southern Blot: used to identify which clones in a library contain a given DNA sequence and to characterize the size of fragments- Northern blot analysis: RNA blotting, shows gene expression- Western blotting: protein blotting, antibody for specific protein- Dideoxynucleotide chain-termination sequencing (sanger sequencing): a double stranded DNa molecule whose sequence is to be determined is converted to single strands that are used as a template for synthesizing a series of complementary strands, then mixed with a primer that is complementary to the target DNA- Computer-automated DNA sequencing uses the sanger method and a computer which was faster than doing it alone- Next generation sequencing (NGS) technology: started in 2005 and started a goal to find new sequencing methods, greatly increase sequencing productivity and decreased sequencing costso Roche 454 sequencing technology: binds DNA fragments to
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