Help
Options
Didn't know it?
click below
click below
Knew it?
click below
click below
Don't Know
Remaining cards (0)
Know
retry
shuffle
restart
0:00
Embed Code - If you would like this activity on your web page, copy the script below and paste it into your web page.
Normal Size Small Size show me how
Normal Size Small Size show me how
BSC 3403
Exam 3
| Term | Definition |
|---|---|
| The general protocol for purifying DNA involves | disrupting cells, centrifuging the lysate to separate the various cellular fractions, adding proteinase K to degrade all of the proteins (including nucleases that can digest the DNA), and using various salts and organic solvents |
| EDTA | chelates metals and inhibits DNases |
| phenol-chloroform extraction, | uses phenol (contains DNA and RNA), chloroform (contains proteins and debris), and isoamyl alcohol (to reduce foaming) to extract the DNA from the impurities; produces hazardous waste and any residual organic solvents will inhibit downstream steps |
| Alkaline lysis | uses SDS to break up phospholipid bilayer and NaOH to dissolve structure proteins. |
| Silica-based methods | allow the selective absorption of DNA to beads in the presence of chaotropic salts. |
| miniprep | based on the alkaline lysis and also involves a silica-based column that will selectively bind DNA; goal is to lyse and remove everything except plasmid DNA |
| An A260 of 1.0 = | 50 μg/mL of DNA in the sample. |
| A DNA sample with an A260/A280 ratio of | greater than 1.5 is considered to be relatively pure. |
| RNA is much less stable than DNA due to | its 2’ hydroxyl group that promotes degradation, and because of this RNases do not require metals and are therefore not inhibited by EDTA; RNA can withstand autoclaving |
| guanidinium isothiocynanate | denatures proteins and inactivates intracellular RNases |
| mRNA | has a polyA tail that can be used to purify via a polyT column; accounts for less than 5% of the RNA in the cell |
| agarose gel electrophoresis | very similar to SDS-PAGE; advantage is ease of preparation, DNA already has a built in negative charge so SDS is not needed; forms a thick stable gel; does not require a stacking gel; samples are loaded perp. to the current; can be a purification step |
| conformation of DNA and gel electrophoresis | supercoiled DNA will usually run more quickly than linear DNA (depending on the degree of supercoiling), and open circular DNA (or “nicked” DNA, plasmids with only a single ssDNA cut) will run the slowest. |
| pulse field gel electrophoresis | For very large DNA samples (>5000 bp); varies the current direction from multiple electrodes to increase sample separation. |
| UV shadowing | involves placing the gel directly on a UV; DNA, which absorbs at 280 nm, should appear as shadows in the gel; Unfortunately, a large amount of DNA is required to see any bands. |
| ethidium bromide | intercalates DNA, can be incorporated directly into the gel prior to polymerization, or gels can be soaked in it after electrophoresis; easiest and most reliable way to visualize DN; nanogram detection; carcinogen |
| SYBR green or methylene blue | Both bind to DNA like ethidium bromide, and are safer to use; These dyes do not offer the same intensity as EtBr when visualizing DNA and can only detect in the microgram range unless optimized. |
| autoradiography | radioisotopes like 32P are incorporated into the DNA and detected with X-ray film; This is a time consuming process and requires the use of radioactive molecules and special film developers |
| freeze squeeze | the band from a gel can be frozen and squeezed in parafilm to remove the agarose form the DNA sample |
| Genome | the complete set of the nucleic acids containing the genes for an organism |
| proteinase K | degrades most proteins (especially nucleases), and is active in SDS, EDTA, urea, and wide ranges of pH |
| The migration distance of fragments in the gel | will be inversely proportional to the log of the number of base pairs |
| Recombinant DNA technology | term for combining DNA sequences that aren’t found together, which is very useful since it allows for the study of genes on a smaller, more controllable scale; process involves the biochemical manipulation of genes using enzymes |
| restriction enzymes | DNA-cutting enzymes; responsible for bacteriophage restriction; digest it at positions where adenines are methylated (Types I and III), and others at specific DNA sequences (Type II), which are more useful; used by bacteria as defense; named by source |
| Palindromic DNA | DNA that can bind to the complement of itself. |
| DNA methylases | naturally present in the cell and are used to determine the original vs. new strand during DNA replication; This methylation will inhibit DNA digestion by restriction enzymes, and protect the E. coli from its own enzymes |
| isoschizomers | restriction enzymes from different species that digest at the exact same sequences and in the same manner as other restriction enzymes from other species |
| neoschizomers | Others may recognize the same sequence of DNA, but cut in a different fashion |
| BSA | helps to stabilize and improve the efficiency of some enzymes as well as prevent the enzymes from sticking to pipettes and microcentrifuge tubes |
| Restriction enzyme tables | useful in determining if two enzymes have compatible buffers and can be used at the same time – if not the fragment must be digested with one, run on a gel, gel extracted, and digested with the other. |
| star activity | when a restriction enzyme is not always site-specific and can cut at an incorrect sequence; rare but increases if the reaction is allowed to proceed for too long or in an incorrect buffer |
| DNA polymerase | responsible for copying DNA during synthesis in the 5’→3’ direction, and making repairs upon DNA damage; can fill in sticky ends to produce blunt ends; requires magnesium |
| E. coli polymerase I and III | can be used to copy DNA. |
| TAQ polymerase | stable at high temperatures and used in PCR; very fast; lacks proofreading; adds A overhangs; makes mistakes |
| Pfu | outstanding proofreading capabilities; slow; rarely makes mistakes |
| exonuclease activity | is beneficial in the 5'-3' direction because it digests DNA and removes downstream primers; however this can cause problems by nonspecifically digesting DNA; 3'-5' direction is the proofreading domain |
| T4 DNA polymerase | can be used to fill in 5’ overhands via standard DNA synthesis, or chew back 3’ overhands with its 3’→5’exonuclease domain. |
| for ligase to function | it must have two pieces of DNA that contain either blunt ends or overhangs that are complementary to one another. |
| sticky end vs. blunt end | Overhang “sticky” ligations are much more efficient than blunt end ligations because the base pairing of the overhands increases the chances of the two pieces of DNA finding each other and sticking together in solution. |
| Topoisomerase | can be used to ligate DNA; relieves tension during DNA replication by breaking the phosphodiester backbone of DNA, passing one strand across another, and reforming the bond. |
| restriction mapping | The process of determining the locations of restriction endonuclease cleavage sites within a piece of DNA |
| cloning | placing a gene of interest into a plasmid, getting the plasmid into a bacterial host, and growing millions of clones of the original bacterial cell that took up the plasmid. |
| transformation | the process of taking up DNA from the environment; inefficient process, and very few cells actually take up DNA in an experiment, but only one cells needs to take up the vector as it can produce millions of copies of itself. |
| using CaCl2 to make competent cells | The calcium ions help to neutralize the DNA and the chlorine ions cause the bacterial cell to swell and create pores; The cells are mixed with the plasmid and heat shocked at 42°C for a short time, and chilled on ice; forces the plasmid into the cell. |
| Electroporation | shocks the cells with an electric current, causing the membrane to permeabilize and take up the plasmid; takes only seconds and gives a time constant that tells the researcher whether the transformation was likely successful or not; expensive, more prep |
| in vitro packaging | using bacteriophages to deliver DNA via transduction into E. coli |
| A replicon | a piece of DNA that is able to function like a chromosome in vivo and is capable of being replicated and partitioned stably over many generations. |
| Vectors (or plasmids or constructs) | dsDNA that are independent from the bacterial chromosome and have the ability to be passed from one generation to the next, and some can replicate in various host organisms; can be designed for cloning or protein expression; limited to under 50,000 bp |
| to be successful, vectors must have | an origin of replication (ori), a multiple cloning site (polylinker) that contains the gene of interest, a selectable marker, and some species require a partitioning sequence, such as the centromere in eukaryotes. |
| origin of replication (ori) | each chromosome must contain this in order be replicated and maintained stably in an organism |
| multiple cloning site (MCS); polylinker | designed to contain several unique sites for restriction enzymes located next to each other; |
| two enzymes with different cut sites is advantageous | because this prevents the plasmid from reannealing (and thus producing false positives), increases ligation efficiency due to the complementary overhangs base pairing, and ensures directionality of the insert during the ligation step. |
| blunt end digestions offer | versatility though, in that two different enzyme digests can be ligated together, and overhang digestions can be filled in or chewed back to blunt ends and ligated together; less efficient |
| selectable marker | ensures that the bacteria that grow are only those that have taken up and maintained the vector, and any non-transformants will die. |
| a standard cloning vector | designed for efficient uptake and replication in the host cell |
| a shuttle vector | can replicate in multiple hosts |
| an expression vector | allows for high levels of protein expression. |
| bacteriophages | can be packaged with the DNA of interest and mixed with E. coli. |
| Cosmids | combination of a plasmid and a lambda phage (and contain cos sites used for phage packaging), and are capable of replicating as a phage, in E. coli, and in eukaryotes; 50,000 bp and useful for making gene libraries |
| Bacterial artificial chromosomes and yeast artificial chromosomes | developed to carry hundreds of thousands or millions of base pairs of DNA. |
| Retroviruses | viral RNA is converted into DNA by the viral reverse transcriptase and is then integrated into the host genome, and any foreign or mutated host gene inserted into the retroviral genome will be integrated into the host chromosome as well; gene therapy |
| phenotypic screening | when a plasmid or cloned gene changes the phenotype of the cell in an obvious way; does not guarantee that the cells are also carrying the gene of interest; good initial screening |
| Conditional mutants | mutants that can grow under one set (permissive) of environmental conditions but cannot grow under different conditions (restrictive or nonpermisive). |
| Conditional lethal mutants | mutants that can grow under one set (permissive) of environmental conditions but die under different (restrictive or nonpermissive) conditions |
| Non-conditional mutants | display the mutant phenotype under all conditions, and require a supplement to survive; |
| A gene library | a collection of all of the genes in an organism and it may or may not include introns or intergenic DNA. |
| A genomic library | contains all of the DNA of a cell in fragments |
| A cDNA library | contains the entire coding sequence and lacks introns and upstream regulatory regions |
| A cDNA subtraction library | can be used to study differences between two organisms |
| Probes | 32P- or fluorescently-labeled ssDNA that can hybridize (base pair) to a piece of ssDNA in a sample and tell you if that sequence is absent or present. |
| dot blot | The most basic application of a probe is to determine simply whether a given sequence is present or not in a sample of DNA; similar to ELISA; tells nothing of size |
| fluorescence in situ hybridization (FISH) | Probes can also be used to determine a gene’s locus on a chromosome |
| A Southern blot | a technique that not only tells you whether a piece of DNA is present or absent, but it also tells you the size of the DNA; similar to western blot; |
| Restriction fragment length polymorphism (RFLP) | allows one to identify whether an individual has a certain gene, or a different/mutant of the gene; needs a restriction enzyme specific to the polymorphic site; can be used to study recombination/crossing over |
| Variable Number of Tandem Repeats (VNTR) | relies on the fact that DNA can slip during replication or damage in repeat regions and this goes unnoticed by DNA polymerase and repair enzymes because the DNA still base pairs properly; replaced RFLP |
| northern blot | almost identical to a Southern blot, except instead of searching for DNA one is searching for RNA, and a labeled DNA or RNA probe is used. |
| RNase Protection Assay | a technique where a labeled probe is hybridized to mRNA creating dsRNA, and an RNase is used to chew up any remaining ssRNA; dsRNA is run on a gel and detected via autoradiography |
| DNA polymerase can only synthesize new DNA | in the 5’→3’ direction, reading the template in the 3’→5’ direction |
| Polymerase Chain Reaction, or PCR | allows for exponential production of a piece of dsDNA |
| Watson strand = | strand on top |
| Crick strand = | strand on bottom |
| The forward primer | is identical to a section of the Watson strand and will bind to the Crick strand. |
| the reverse primer | is identical to a section of the Crick strand and will bind to the Watson strand. |
| primers | generally 20-30 nucleotides in length and should have greater than 50% GC content to improve bind to the template; bind to DNA between 100 and 1000 base pairs apart, facing one another. |
| melting temperature (Tm) | This is the temperature at which 50% of the primer is bound to the template DNA, and is determined by the number of AT and GC base pairs in the primer; can be estimated by adding 2°C for every AT pair and 4°C for every GC pair |
| annealing temperature | should be ~5°C lower than the lowest Tm of a pair of primers; too high causes the primer to not bind well and no copying; too low causes the primer to bind nonspecifically |
| quantitative PCR, or qPCR (also called real-time PCR or RT-PCR because samples are measured in real time) | measure the kinetics of the PCR reaction in its logarithmic phase; sensitive enough to detect 2-fold differences between samples; simultaneously quantify and amplify DNA |
| fluorescent resonance energy transfer (FRET) | that involves the transfer of energy between a donor and acceptor molecule; When the donor and acceptor are close in proximity, a transfer of energy occurs; when the two are far apart, no transfer occurs |
| SYBR green dye | fluoresces when bound to dsDNA, but not ssDNA; the advantage of being usable with any sample; disadvantage is that it will bind to any dsDNA, while probes are specific to a predetermined target and provide a permanent record of amplification; melt curve |
| melt curve | achieved by raising the temperature of the qPCR products slowly and measuring the change in fluorescence as the DNA dissociates; Multiple peaks indicate multiple samples of DNA; Peaks at lower temperatures indicate primer dimers or other contaminants |
| Ct threshold line | the point at which you (or likely computer software) choose to compare samples; can be used to compare samples to each other and determine the relative amount of starting material in the original samples |
| reverse transcriptase PCR, or RT-PCR (don’t confuse this with real time PCR) | uses reverse transcriptase, an RNA-dependent DNA polymerase, to synthesize DNA from an mRNA template; very sensitive and low amounts of starting mRNA needed; |
| reverse transcriptase quantitative PCR, or RT-qPCR (or RT RT-PCR). | the combination of qPCR and RT-PCR; replaced northern blotting for determining cellular mRNA |
| TA cloning | PCR products with A overhangs are directly ligated into a linearized vector containing T overhangs; avoids the need for a restriction enzyme digestion and gel extraction on your PCR products; does not guarantee insert directionality. |
| site-directed mutagenesis | uses traditional PCR and various primers to produce alterations in a gene product; requires two pairs of primers; |
| multiplex PCR | use more sets of primers and amplify multiple genes of interest; complex and time-consuming procedure that requires much optimization; |
| Touchdown PCR | involves starting PCR at a high, non-permissive annealing temperature and then incrementally lowering the annealing temperature in subsequent rounds; At some point, the primer with the highest specificity will bind and the target will be amplified |
| Nested PCR | increases the specificity of PCR by reducing nonspecific product amplification; uses four primers; primers 1 and 2 may have amplified a nonspecific product, primers 3 and 4 should only bind and amplify the gene of interest. |
| gene typing | allows a scientist to identify an organism’s genotype. |
| forensic DNA typing | has replaced RFLP and VNTR analysis in paternity testing and crime scene analysis because PCR is straightforward and requires very little starting sample; uses short tandem repeat polymorphisms that are highly variable to characterize various gene loci. |
| Amplified Fragment Length Polymorphism PCR (AFLP PCR) | circumvents the problem of not having an external primer to amplify a fragment of DNA by cutting DNA with restriction enzymes and ligating on adapter molecules; more sensitive, has higher reproducibility, can amplify up to 100 fragments at a time |
| Random Amplification of Polymorphic DNA (RAPD PCR) | uses short primers (~6 nucleotides) as priming sites for random DNA polymerase synthesis |
| ancient DNA | Modern techniques of DNA extraction permit the isolation of exceedingly tiny amounts of DNA; difficult to clone because of its very low quantity, contamination, and covalent modifications |
| asymmetric PCR | generates ssDNA probes via an asymmetrical primer ratio; useful for DNA hybridization and 32P-labeled nucleotides can be used |
| anchored PCR | allows for the amplification of gene when only part of the sequence is known and part is unknown or variable |
| Solid-state PCR | performs PCR in oil (emulsion PCR) or on beads (bridge PCR), from which DNA is easily retrieved; reduces cross-contamination, and does not require gel electrophoresis or cloning. |
| Helicase-dependent PCR | uses the enzyme helicase (as opposed to a thermocycler) to separate the two complementary DNA strands just as it occurs in the cell, and at lower temperatures than needed in traditional PCR; faster but requires more template DNA and optimization |
| Long PCR | PCR is limited to copying several thousands base pairs, and this can be overcome in some cases by using TAQ combined with Pfu and longer extension times |
| inverse PCR | can be used to amplify regions of DNA of unknown sequence that flank a known sequence |
| Maxam-Gilbert Sequencing | using a radiolabeled 5’ phosphate and 3’ cleavage of DNA with various chemicals; could be performed directly on DNA without the need for cloning or ssDNA; complex and time consuming; read bottom to top |
| Sanger Sequencing (or chain termination sequencing) | involves using dideoxy nucleotides (ddNTPS) that lack a 3’ hydroxyl group to terminate DNA synthesis; requires an acrylamide gel which can be read to obtain the DNA sequence (reverse and complement from bottom to top); 500-1000bp |
| Pyrosequencing (454 sequencing) | can sequence hundreds of megabases within hours by binding small fragments of DNA to beads immersed in oil and amplifying them via emulsion PCR. |
| Ilumina (Solexa) sequencing | uses bridge PCR to link primers to a solid support medium; As each base is added, it is detected with fluorescence |
| Ion semiconductor sequencing (or ion torrent sequencing) | works by adding dNTPs one at a time to the DNA to be sequenced; This changes the pH which is detected by a machine |
| Nanopore sequencing | does not directly measure fluorescence or change in pH involves passing a DNA sequence through a small nanopore and measuring the change in current; very fast |
| Microarray chips | contain millions of DNA oligonucleotides of various sequences; allows for very fast and efficient sequencing of large pieces of DNA, but is currently limited to sequencing only short fragments of DNA; if a spot lights up, the sequence is present |
| Mass spectrometry | used to sequence DNA since each nucleotide has a slightly different molecular weight; very fast but is currently limited to about 100 nucleotides |
Created by:
JacobGant
Popular Biology sets