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UKCD Biochem Lec 5

terms from Biochem lecture 5

Lecture 5 Terms Biochemistry
Restriction Enzymes aka restriction endonucleases split DNA into specific fragments, recognize specific base sequences in DNA cleaving both strands at specific points, only found in prokaryotes where they cleave foreign DNA, own DNA protected by methylation
Palindromic cleavage sites of restriction enzymes that possess two fold rotational symmetry
Restriction Fragments fragments of DNA cleaved by restriction enzymes
Gel Electrophoresis separation of restriction fragment using agarose or acrylamide gels of varying porosity, can be done since DNA is negatively charged due to the phosphate
Southern Blotting restriction fragments containing a specific sequence can be identified using a labeled complementary DNA strand after gel electrophoresis transferred to nitrocellulose label DNA probe w/ complementary fragments reveal positions of complementary fragments
Southern Blotting used to identify specific DNA fragments
Northern Blotting used to identify specific RNA fragments
Western Blotting used to identify specific proteins using antibodies
Sanger Dideoxy Method method of DNA sequencing
Controlled Interruption of Enzymatic Replication DNA sequencing of fragments of DNA, requires primer and DNA polymerase, use radioactively labeled dexoyribonucleoside triphosphates , small amount of 2’,3’-dideoxy analog of 1 of 4, each time incorporated replication stops giving fragments w/ known end
DNA Sequencing Now uses fluorescent labels attached to primer, different color for 4 bases similar method as before
Human Genome over 99% similar to rodents, contains 3 billion base paints but only 30,000 genes the remaining is repetitive elements, inactivated viruses, pseudo genes, control elements which all equal our evolutionary history
Polymerase Chain Reaction (PCR) amplifying specific DNA sequences, target sequence amplified if flanking sequences known, can be repeated many times just by changing the temperature of same one solution, all new strands act as templates, after n cycles target sequence amplified 2n fold
3 Steps of PCR 1. Strand separation 2. Hybridization of primers 3. DNA synthesis
PCR Strand Seperation two strands of parents DNA separated by heating to 95c for 15 s in presence of excess primers
PCR Hybridization of Primers solution is abruptly cooled to 54C to allow each primer to hybridize to DNA strand, primers present in excess prevents parent duplex from reforming
PCR DNA Synthesis solution is heated to 72C the optimal temperature for Taq DNA polymerase from Thermus aquaticus, polymerization allowed for 30 seconds synthesizing copies of both strands
PCR in medicine readily detect bacteria and virus, can find tiny amounts before infection is evident
PCR in forensics “DNA fingerprinting” 13 genomic regions unique to individuals, changes of 2 individuals sharing all 13 is 1 in 109
Cloned amplified many times can happen to genes
Splicing attaching DNA fragment
DNA vector what spliced DNA is attached to , replicate autonomously in appropriate host requires restriction enzymes and DNA ligase
Recombinant DNA prepare vector by cleaving at specific restriction enzyme site, cohesive or sticky ends created allow DNA fragment with same ends to be inserted, joined by DNA ligase forming phophodiester bond at break in DNA chain
Plasmids and Phages vectors of choice for cloning in bacteria
Plasmids circular duplex DNA molecules that occur naturally in some bacteria, modified to carry various useful genes and handy restriction sites when used to create recombinant DNA
Accessory Chromosomes replication independently of host chromosome, required by plasmids and phages
pUC18 useful plasmid, basis for many specialized vectors, carries genes for resistance to ampicillin and beta-galactosidase, multiple restriction sites allow insertion of genes which inactivates beta gal allows detection blue-none, white-insertion of gene
λ phage useful bacteriophage (bacterial virus) vector, if uses lytic pathway, viral genes express causes death of host, if uses lysogenic pathway phage DNA inserted and replicated along with host DNA, pathway used determined by environmental factors,
Mutant λ phage constructed for cloning purposes including λ gt- λ Beta contains only two EcoRI sites rather than 5 allowing middle 25% of DNA to be removed and suitably long foreign inserted and can then be used to infect bacteria for cloning purposes
Cloning from Digests digest of genomic DNA, either mechanically or restriction enzymes, random process results in overlapping separated by gel electrophoresis, once isolated synthetic linkers added, cohesive ends are created w/ restriction enzymes, inserted into phage vector
Lysate resulting fragments of human DNA hosted in large virus particles where nearly entire genome represented caused by cloning from digests
Genomic Library resulting lysate from cloning digest
DNA Hybridization recombinant phage placed on lawn of bacteria, infect bacteria-plaque which contains identical phage, replica made w/ nitrocellulose sheet, treated w/ NaOH which creates denatured DNA then labeled with specific probe and complementary DNA will be detected
Yeast Artifical Chromosomes (YACs) allow cloning of very large genes containing introns, replicated in yeast, contain autonomous replication sequence where replication beings, two telomeres or normal ends of eukaryotic chromosomes, selectable marker gene and a cloning site
Reverse Transcriptase synthesizes DNA complementary to RNA template when provided DNA primer base paired to RNA, poly(T) primer used, new primer added and terminal transferase adds nucleotides to 3’ end, synthetic linkers added, cohesive ends formed so can insert into vector
Expression varies widely between different cell types as indicated by mRNA quantities, also varies as a function of cell type and as cells respond to changes in physiological circumstances
Complementary DNA (cDNA) results from reverse transcriptase, mRNA in a cell
DNA Microarrays/Chips way to analyze gene expression, constructed by fixing oligonucleotides to chip complementary to part of specific gene w/known position, fluorescently labeled cDNA library hybridized to support and differences in fluorescence -expression levels
Postranlational modification common in eukaryotic proteins but not in bacteria due to lack of correct enzymes therefore eukaryotic genes are best expressed in eukaryotic systems, therefore there are many ways to insert recombinant DNA into animal cells
Foreign DNA precipitated by calcium phosphate taken up by animal cells, inexpensive but low efficiency
DNA microinjected into cells DNA injected into nucleus through fine tipped glass micropipette, resulting in 2% viable cells
Anionic liposomes coated with foreign DNA fused with cells, high efficiency, cell type dependent efficiency and expensive
Tiny Tungsten particles coated with foreign DNA fried into cells with gene gun good for penetrating tough plants
Viral Vectors defective viruses that cannot reproduce are engineered to carry the gene of interest, retroviruses require dividing cell types, Adeno-associated virus can also infect non-dividing cell types
Transgenic Animals introducing foreign genes on recombinant DNA into fertilized egg ex giant mice
Knock Out/Gene disruption function of gene can be probed by inactivating it and looking for consequences
Homologous Recombination relied on by gene disruption, regions of DNA with strong sequence similarity can exchange segments, therefore if gene sequence is known, inactive form crated and inserted into cell therefore inactivating cellular gene
RNA Interference aka RNAi siRNA double stranded RNA is interpreted by eukaryotic cells as foreign, inducing degradative response, dicer cleaves dsRNA into pieces, strands separated, each is incorporated into separate RNA-induced silencing complex, specifically cleave mRNAs complementary
Viral Transduction of Genes many viruses only infect dividing cells, cancer studies Adenoassociated virus (AAV) and Lentivirus infect non-dividers, capsid (cap) specifies for receptor for virus, promoter choice can direct cell type-spp expression
Site Specific Mutagenesis recombinant DNA makes it possible to introduce specific changes into a protein’s amino acid sequence including insertions, deletions, substitutions and designer genes
Deletion cleaving a plasmid at two sites and religating to form a smaller circle, usually moves a fairly large block of DNA sequence, smaller deletions are created by cleaving at a single site and then removing single bases using an exonuclease and religating
Transposon mediated “jumping genes” deletion can be performed in intact cells or organisms
Substitute used in error prone PCR to randomly substitute nucleotides in a region bounded by primers
Oligonucleotide-Directed Mutagenesis single amino acid substitutions
A specific substitution can be made if you have the gene for the protein, cDNA in plasmid, or known base sequence around the site of substitution
Site Specific Mutagenesis synthetic oligonucletide primer complementary to gene prepared, 2 strands separated, primer hybridized, mismatches created are intolerable, primer elongated, double stranded circle is closed, replication creates 1 w/ mutation and 1 w/out
Cassette Mutagenesis variation on DNA where a short sequence is substituted into a plasmid, resulting in a mutant sequence being inserted into the protein product
Designer Genes aka novel proteins, can be created by splicing together gene segments that encode protein domains
Created by: wiechartm



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