Term
| who discovered restriction enzymes? |
|
Definition
| Swiss microbiologist Werner Arber and Stuart Linn |
|
|
Term
| what did Werner Arber's daughter Sylvia call restriction enzymes? |
|
Definition
|
|
Term
| Arber discovered restriction enzymes while... |
|
Definition
| studying a phenomenon known as host-controlled restriction of bacteriophages (also known at the time as host-controlled modification of bacteriophages). |
|
|
Term
|
Definition
| viral particles that invade bacteria and replicate their own DNA independently of the bacterial chromosomal DNA |
|
|
Term
| the basis for Arber discovering restriction enzymes |
|
Definition
| he wanted to know why bacterial phages did better in some strains of bacteria than others |
|
|
Term
| what Arber proposed before discovering restriction enzymes |
|
Definition
he theorized that previous exposure to a particular bacterial strain somehow protected that phage from that bacterial strain
Arber also proposed that there were specific sites in the virus with unmodified genome where restriction occured |
|
|
Term
| Arber and Linn referred to the enzyme responsible for this "endonucleolytic scission" as... |
|
Definition
| endonuclease R, a name later changed to EcoB |
|
|
Term
| a restriction enzyme discovered in E. coli after Arber and Linn's research |
|
Definition
|
|
Term
|
Definition
| enzyme discovered in another bacterial species that degrades foreign phage DNA but not the bacterial host's DNA |
|
|
Term
| restriction or recognition site |
|
Definition
| the actual nucleotide sequence of the specific site where a restriction enzyme cleaves |
|
|
Term
| significance of discovering restriction or recognition site |
|
Definition
| confirms Arber's hypothesis that restriction enzymes are extremely selective with regard to where they make their cuts |
|
|
Term
| how many restriction enzymes are there? |
|
Definition
|
|
Term
| how many restriction sites are there? |
|
Definition
|
|
Term
| length of restriction sites |
|
Definition
|
|
Term
| most restriction sites are palindromic, meaning... |
|
Definition
| the sequence reads the same forward and backward |
|
|
Term
| how bacterial enzymes are named |
|
Definition
| first 3 letters of bacterium from which it was isolated, e.g., Eco for E. coli, fourth letter for particular strain, e.g., EcoB, and Roman neumerals for enzymes from the same strain, e.g., EcoBII |
|
|
Term
| the 3 categories of restriction enzymes recognized today |
|
Definition
|
|
Term
| type I restriction enzymes |
|
Definition
| they recognize specific DNA sequences but make their cut at seemingly random sites that can be as far as 1,000 base pairs away from the recognition site |
|
|
Term
| type II restriction enzymes |
|
Definition
| they recognize and cut directly within the recognition site |
|
|
Term
| type III restriction enzymes |
|
Definition
| they recognize specific sequences but make their cut at a different specific location that is usually within about 25 base pairs of the recognition site |
|
|
Term
| purpose of restriction enzymes |
|
Definition
| defense against invading viruses |
|
|
Term
| how bacteria protect their DNA |
|
Definition
| by modifying their own recognition sequences |
|
|
Term
| how bacteria modify their own recognition sequences |
|
Definition
| usually by adding methyl (CH3) molecules to nucleotides in the recognition sequences and then relying on the restriction enzymes' capacity to recognize and cleave only unmethylated recognition sequences |
|
|
Term
| how bacteriophages protect theirselves from restriction enzymes |
|
Definition
| bacteriophages that have previously replicated in a particular host bacterial strain and survived are similarly modified with methyl-labeled nucleotides and thereby protected from cleavage within that same strain |
|
|
Term
| the first major application of restriction enzymes |
|
Definition
| as a tool for cutting DNA into fragments in ways that would make it easier to study and, in particular, identify and characterize genes |
|
|
Term
| a second major use for restriction enzymes |
|
Definition
| as a device for recombining, or joining, DNA molecules from different genomes |
|
|
Term
| the goal of recombining, or joining, DNA molecules from different genomes |
|
Definition
| identifying and characterizing a gene or studying gene expression and regulation |
|
|
Term
| how scientists today separate DNA fragments |
|
Definition
| restriction enzyme digestion, followed by electrophoresis |
|
|
Term
|
Definition
| a DNA or RNA molecule with a base sequence that is complementary to a DNA sequence of interest |
|
|
Term
|
Definition
| to identify where in the genome (i.e., on which fragment) the sequence of interest is located |
|
|
Term
|
Definition
1: DNA fragments electrophoretically separated
2: fragments transferred from gel to solid medium, or membrane |
|
|
Term
| what is done to the DNA fragments after Southern blotting? |
|
Definition
3: probes are used to bind to the sequences of interest
4: membrane is washed to leave only the probes bound to the membrane |
|
|
Term
|
Definition
| technique used to detect radioactive probes after the membrane is washed |
|
|
Term
|
Definition
| DNA fragments with no sticky ends |
|
|
Term
|
Definition
| overhanging single strands of DNA on a DNA fragment |
|
|
Term
|
Definition
| enzyme that can covalently bind complementary sticky-end fragments |
|
|
Term
| what the utilization of sticky ends and DNA ligase enables molecular biologists to do |
|
Definition
| create seemingly limitless combinations of recombinant DNA |
|
|
Term
|
Definition
1: near-boiling temperature causes double-stranded DNA to separate, or denature into single strands
2: temperature is decreased and short DNA sequences known as primers bind, or anneal, to complementary matches on the target DNA sequence. The primers bracket the target sequence to be copied
3: At a slightly higher temperature, Taq polymerase binds to the primed sequences and adds nucleotides to extend the second strand. This completes the first cycle.
4: repeat 1-3 |
|
|
Term
|
Definition
| circular pieces of DNA that replicate independently of the host's chromosomal DNA |
|
|
Term
|
Definition
mainly bacteria, but can also exist in...
-archaea
-eukaryotes |
|
|
Term
| some eukaryotes that can have plasmids |
|
Definition
|
|
Term
| some beneficial functions plasmids can provide to the host |
|
Definition
-resistance to antibiotics
-degradative functions
-virulence |
|
|
Term
| what natural plasmids contain |
|
Definition
-origin of replication (all have this)
-typically include a gene that is advantageous for survival |
|
|
Term
| what artificial plasmids used in the lab are used for |
|
Definition
| to introduce foreign DNA into the cell |
|
|
Term
| minimally, plasmids made in the lab contain... |
|
Definition
-origin of replication
-selection marker
-cloning site |
|
|
Term
| what makes plasmids attractive tools for life scientists and bioengineers? |
|
Definition
| The ease of modifying plasmids and the ability of plasmids to self-replicate within a cell |
|
|
Term
|
Definition
|
|
Term
| some vector elements within a plasmid |
|
Definition
-Origin of Replication (ORI)
-Antibiotic Resistance Gene
-Multiple Cloning Site (MCS)
-Insert
-Promoter Region
-Selectable Marker
-Primer Binding Site |
|
|
Term
| Origin of Replication (ORI) |
|
Definition
| DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins |
|
|
Term
| Antibiotic Resistance Gene |
|
Definition
| Allows for selection of plasmid-containing bacteria. |
|
|
Term
| Multiple Cloning Site (MCS) |
|
Definition
| Short segment of DNA which contains several restriction sites allowing for the easy insertion of DNA. In expression plasmids, the MCS is often downstream from a promoter. |
|
|
Term
|
Definition
| Gene, promoter or other DNA fragment cloned into the MCS for further study. |
|
|
Term
|
Definition
| Drives transcription of the target gene. Vital component for expression vectors: determines which cell types the gene is expressed in and amount of recombinant protein obtained. |
|
|
Term
|
Definition
| The antibiotic resistance gene allows for selection in bacteria. However, many plasmids also have selectable markers for use in other cell types. |
|
|
Term
|
Definition
| A short single-stranded DNA sequence used as an initiation point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids. |
|
|
Term
| Due to their aritificial nature, lab plasmids are commonly referred to as... |
|
Definition
| “vectors” or “constructs” |
|
|
Term
| what scientists may do to insert a gene of interest into a vector |
|
Definition
| utilize one of a variety of cloning methods (Restriction enzyme, Ligation indepenent, Gateway, Gibson, etc) |
|
|
Term
|
Definition
-Restriction enzyme
-Ligation indepenent
-Gateway
-Gibson
-etc. |
|
|
Term
| how the cloning method is chosen |
|
Definition
| based on the plasmid you want to clone into |
|
|
Term
| Addgene’s Plasmid Guide includes information about... |
|
Definition
-molecular cloning
-how to choose a plasmid vector
-molecular biology tools and references
-how to maintain your plasmid stocks
-multiple protocols and troubleshooting tips to make plasmid usage as simple and straightforward as possible |
|
|
Term
| Generally, scientists use plasmids to... |
|
Definition
| manipulate gene expression in target cells |
|
|
Term
| some characteristics of plasmids that enable molecular biologists to broadly utilize plasmids across a wide range of applications |
|
Definition
-flexibility
-versatility
-safety
-cost-effectiveness |
|
|
Term
| Some common plasmid types |
|
Definition
-Cloning plasmids
-Expression plasmids
-Gene knock-down plasmids
-Reporter plasmids
-Viral plasmids
-Genome engineering plasmids |
|
|
Term
| some things plasmids are being used for |
|
Definition
vectors for experiments encompassing...
-fluorescent imaging
-recombinant DNA technology
-mass protein production
-disease modeling
-drug discovery
-genome editing |
|
|
Term
| 2) Sylvia, the 10 year old (at the time) daughter of Werner Arber said the following, If a foreign king invades a bacterium, this servant can cut him in small fragments. Who is the “king” in which she is referring to? |
|
Definition
|
|
Term
| DNA bands in an agarose gel are visualized by... |
|
Definition
|
|
Term
| the role of loading dye in gel electrophoresis |
|
Definition
| helps you load the sample into the well |
|
|
Term
| are DNA fragments by themselves visible during the gel electrophoresis procedure? |
|
Definition
|
|
Term
| when DNA fragments are visible |
|
Definition
normally after electrophoresis
if stained with EtBr, they become visible when placed on UV; causes EtBr to fluoresce |
|
|
Term
| does intensity of staining correspond to number of DNA fragments? |
|
Definition
|
|
Term
| true or false? Assuming that the two RE digestion reactions that gave the results shown in panel A of Figure 1 contained equal quantities of plasmid DNA. It would be expected that the single band shown in lane 2 would have equal or more intensity than any band shown lane 3. |
|
Definition
|
|
Term
| why the intensity of the top few bands in electrophoresis can decrease from top down |
|
Definition
| because of fewer base pairs in each fragment as you go down |
|
|
Term
|
Definition
|
|
Term
| why is it helpful to know where the restriction sites in a viral genome are? |
|
Definition
-helps you know the size of the expected fragments
-helps you know what % of agarose to run |
|
|
Term
| true or false? The reference ladder can tell you if your gel needs to be run longer by the number of bands present. |
|
Definition
|
|
Term
| what's used to make a plasmid? |
|
Definition
|
|
Term
| what's the building blocks of a plasmid? |
|
Definition
|
|
Term
| does the MCS occur naturally in plasmids? |
|
Definition
|
|
Term
|
Definition
|
|
Term
| what concentration does the loading buffer work best at? |
|
Definition
|
|
Term
| some possible sources of bands in an unsuccessful PCR experiment |
|
Definition
1: starting material DNA
2: excess primers
3: primer-dimers
4: off-target amplification |
|
|
Term
| the types of primer molecules used in PCR rxns |
|
Definition
-forward primers
-reverse primers |
|
|
Term
| what determines the success of a PCR rxn? |
|
Definition
| whether or not you put it thru a correct thermal cycling program |
|
|
