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Molecular Exam 1
Molecular Biology Fall 2012 Exam Flashcards
| Question | Answer |
|---|---|
| Vector | Plasmid or viral DNA fragment clones which contains: 1) Place to put your DNA 2) Selection (normally antibiotic) 3) origin of replication |
| rDNA production by restriction and ligation | use restriction enzymes to cut in a specific place --> recognition site usually 4-8 bp, palindromic; creates blunt or sticky ends |
| Directional Cloning | Use 2+ restriction enzymes to cut to create different compatible ends, allowing control of the direction of DNA insertion. |
| Isoschizomer | Two restriction enzymes with the same recognition and cut site but different names, isolated from different organisms |
| Two main strategies for cloning a DNA fragment | 1) Ligation using restriction enzyme and ligase 2) Recombination - desired DNA fragment is amplified by PCR & inserted into a vector by recombination enzymes by virtue of sequence homology |
| Polymerase Chain Reaction (PCR) | 1) Denaturation and separation of two strands using heat 2) Anneal 2 synthetic oligoNT primers 5' of the target sequence 3) Extend primers using DNA polymerase **can add RE sites to the end of primers so they are included in the final product |
| Selection vs. Secondary Selection | Choose cells with just the plasmid DNA vs. choose plasmids with DNA insert only (ex. blue / white screening) |
| Cloning / rRNA creation by Recombination | -Amplify DNA sequence using PCR and add recombination sites to primer on 5' end and also sequences complementary to 3' end so amplified DNA contains vector sequences at its end - Use recombinase enzymes to directionally insert PCR product to vector DNA |
| Gateway cloning | Shuttle DNA fragments between different cloning vectors - Combine DNA fragments from PCR + cDNA library + r.e. digest & ligation |
| Libraries | A) Genomic - all DNA in an organism - from RE digests B) cDNA - tissue / time / condition specific but includes all mRNA for that condition - created via reverse transcription C) Plasmids not large enough = phage-based vectors up to 50kb |
| Reverse Transcription | Creation of ssDNA from RNA template 1)use oligo-dT to bind polyA tail (eukaryotes) and reverse transcriptase for 1st strand 2) RNAse to destroy RNA 3) DNA polymerase to synthesize first strand 4) |
| Site-Directed Mutagenesis (works for bacteria and as part of PCR amplification process) | alters DNA sequences & allows functional testing - use PCR primers w/ mutated base --> PCR whole molecule using very accurate T4 DNA polymerase --> after many rounds most molecules mutated --> retransform into bacteria --> test colonies. |
| Forward Genetics | phenotype --> gene random mutagensis via chemical mutagens (EMS/MMS) and UV mutagenesis (XRT) *hard to find gene after mutagenesis seek to ID gene involved in causing an observed phenotype or disease |
| Reverse Genetics | gene --> phenotype specific mutagenesis gene already in hand; seek to understand that gene by altering sequence & analyzing resulting phenotypic changes in vivo |
| Transposon Mutagenesis (Random Mutagenesis | "natural" sequence flanks recognition sites & transposase seq. is cut & pasted thru genome - in vitro seq. flanks selectable marker- transposase elements separate&inducible - add reporter & integrate near promoter/enhancer to assess gene activity |
| Transposon | discrete DNA element that moves around genome |
| Random Plant Mutagenesis | Use A. tumefaciens plant pathogen which inserts Ti plasmid & thus puts T-DNA into plant chromosome...replace T-DNA w/ gene of interest + selectable marker OR transposon in lab (plasmid has T-DNA, oriC, and virulence factors which give transfer function) |
| Chromophore | Co-factors that cause otherwise colorless proteins to absorb and emit light in the visible spectrum |
| Beer's Law | A = Elc |
| Chromophore | Co-factors that cause otherwise colorless proteins to absorb and emit light in the visible spectrum |
| Beer's Law | A = Elc |
| cell fractionation | A separation of organelles and the cytosol, without damaging the organelles. (UV rays, high pressure, etc) |
| Size-exclusion Chromatography | Separation based on size, the larger proteins are eluted first as the smaller become trapped in the pores of the beads present in the column. (Gel filtration also takes shape into consideration). |
| Ion Exchange Chromatography | Matrix containing a positive or negative charge. Positive surface charge binds to negatively charged cation exchange columns. Bounde proteins are eluted with high salt concentrations. |
| Affinity Chromatography | Separation of proteins based on their binding ability, used in purification. |
| Purification of proteins through tagging | 1) Fusion protein is engineered into a protein (contains tag). 2)The fusion protein is ran through affinity chromatography which is attached to the column containing binding sites for the tag. 3) Fusion protein flushed with molecule that wants the B-sites |
| High performance liquid chromatography | Separates small molecules (peptides, nucleic acids), Reverse - Phase: binding occurs to the hydrophobic alkyl chains and then eluted with organic solvent |
| Thin Layer Chromatography | Separation based on the rate at which molecules travel up an absorbant. Usually on a cilica or cellulose strip. |
| Gel electrophoresis | Made of agarose (several hundred bp - 250000bp) or polyacrylamide (2-700bp). ^ % causes smaller pore size. Voltage used as well as dyes and Ethidium Bromide. |
| Pulse field Gel | 100,000 bp+ Electric pulses reorient the large DNA strands allowing them to reorient and separate even with great size. |
| SDS-Polyacrylamide Gel Electrophoresis (PAGE) | Used to separate proteins. The proteins are broken down with heat and the SDS binds to the hydrophobic regions. This coats them in a negative charge and allows separation to occur. |
| SDS-Polyacrylamide Gel Electrophoresis (PAGE)- SetUp | 1) Discontinues: Low acyrlimide, acidic "stacking gel. Focuses protein into a tight band. 2) High concentration, basic "resolving gel" separates based on size once the proteins are in tight bands due to change in ionization (zwitterion helps) |
| Isoelectric Focusing Gel (IEF) | Protein travel until their pH reaches neutral. The gel has a pH gradient: Loading occurrs at high pH end and migrates towards low pH end. |
| Two-dimensional gel electrophoresis | 1)first dimenstion IEF: separation on pI 2) second dimension SDS-PAGE: separation by size. |
| Sequence of RNA polymers | 1)5' end labelled with radioactive phosphate using T4 polynucleotide kinase and ATP. 3' : pCp using T4 RNA ligase. 2)exposed to nucleases, and put on denaturing PAGE. Ribonuclease T1 cleaves after G residue: U2 after A residue. |
| Chain-termination dideoxy sequencing | 1)oligonucleotide attached to DNA or RNA, then extended with DNA polymerase reverse transcriptase respectively. When NT lacking 3' OH- groups are added extension is terminated. For sequencing four are run each with one of the 4 dNTP's and some 2'3' DDNT |
| Chain-termination dideoxy sequencing: Comparison | Random chains are generated at different lengths. The sequence of the DNA being made can be read by comparing the different extension products. Dideoxynucelotides are typically labelled for convenience. |
| Mass Spectrometry | Identifies a molecule based on its mass to charge ratio. Large proteins broken down with proteases and then massed. |
| Tanden Mass Spectroscopy | proteolytic fragments are broken down to amino acids allowing them to be identified. |
| Southern Blot Analysis | DNA fragments cut with specific restriction enzymes and then placed on a gel. Then denatured with alkali to expose single strands and placed on a membrane. Radioactive complementary strand placed to find DNA fragment,and they are annealed. Exposed by Xray |
| Colony Hybridization | 1)Plasmid transformation into bacteria 2)cell culture then nitrocellulose fiber 3)denaturation of the DNA 4)placed on a gel and then exposed to comp. labelled DNA 5)desirable clones identified and removed |
| Flourescent in situ hybridization(FISH) | Cells fixed to slides and DNA hybridized with differently colored (Flour) DNA. Chromosomes are then stained with a dye DAPI. This allows the detection of reciprocal translocation |
| Spectral Karyotyping (SKY) | Each chromosome purified and dyed with a ratio of 5 different flourescent dyes. Pooled, denatured and used to pool a metaphase spread of chromosomes. This allows any differentiation of colors that occcurs on chromosomes to be identified. |
| Chromosome painting | one individual chromosome is purified and dyed |
| Array Comparative Genomic Hybridization | Quantitative method of finding amplifications and deletions. Labelling genomic DNA with a flourophore. The hybridized DNA is then attached to a DNA microarray. Then the normal DNA is also placed in an array and they are overlapped.yellow = normal |
| northern blot analysis | RNAs are separated by size with a denaturing gel - transfered to + charged membrane. The RNA is labelled with probes. These are then used to identify by comparing position of signal in the membrane and the position of marker band of known size. |
| quantitative reverse transcription-polymerase chain reaction (qRT-PCR) | cDNA copy is made from RNA, then the cDNA is amplified by PCR with a 2nd primer in the transcript. Limited to segment of RNA found between the primers. Advantage is it allows very small amounts of RNA to be detected. cDNA made = exp. RNA |
| Nuclease protection assay | A labelled probe is put into solution to anneal to the RNA of interest. The probe has some nucleotides that dont match, S1 nuclease is then introduced to digest the overhanging ends and single stranded nucleic acids. Gel. RNA = undigested probe on gel |
| Primer extension assay | Oligonucleotide - attached complementary to 5' end of RNA molecule. Reverse transcriptase runs and extension falls off when reaching the 5' end. Compare extended product to sequencing ladder. |
| 5' or 3' RACE | 1)Oligonucleotide of known sequence is ligated to 5' end, and then reverse transcribed. 2) cDNA product has extra string of nucleotides at one end. 3)DNA tagged end amplified with two oligo. in PCR. comp. to tag sequence and known segment of RNA. Prod.seq |
| Reporter Genes | Fusion of reporter gene to the regulatory region. This allows one to observe gene expression. An example is B-galactosidase - breaks down galactose for a colored product. |
| Western Blotting | 1)Protein mixture loaded and separated by PAGE and electro. 2)Gel placed on specialized filter that can bind proteins 3)Proteins transfered to filter with electric field 4)Filter exposed to antibody that identifiese with protein of interest |
| Western Blotting (Continued) | 5)The filter is washed to remove any unbound antibody, and then exposed to a second antibody that is conjugated to a detectable molecule.6)The filter was washed and processed for the flourophores attached to the protein of interest. |
| If protein has no antibody | Add protein tag to the N or C-terminus of the protein, and then use an antibody that binds specifically to that tag |
| Co-immunopurification | A protein mixture is exposed to an antibody, and then microbeads are exposed to the mixture and bind to the antibodies as well flushing the protein of interest out of the system. |
| Chromatin Immunoprecipitation (ChIP) | 1)chemical reagent crosslinks DNA to proteins 2)Chromsomal DNA sheared into small fragments 3)antibodies introduced and the proteins are immunoprecipitated 4)reagent added that reverses cross-links 5)DNA undergoes PCR and hybridizes to a microarray |
| Two-Hybrid Analysis | Bait and Prey process |
Created by:
CharanyaKay
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