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25 Cards in this Set
- Front
- Back
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--rDNA history--
Field emerged in... Marmur, isolated DNA from... Aber discovered... Smith, discovered... In 1970’s Cohen, Boyer, and Berg developed |
the 1960’s
E.coli & Serratia marcescens restriction endonucleases HindIII from Haemophilus influenzae grafting of DNA molecules, aka cloning |
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What are enzymes that recognize and cleave specific sequences in dsDNA that exhibit two fold symmetry around a central axis?
How many have been isolated from different bacteria? These enzymes recognize sequences that read the same in opposite directions, otherwise known as BLANK. The DNA gets cleaved into several restriction fragments with single stranded overhangs otherwise known as BLANK BLANK. |
restriction endonucleases
500 different restriction endonucleases palindromes sticky ends |
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EcoRI, recognizes the following sequence:
(Don't memorize) |
5’-PO4 –G-A-A-T-T-C- 3’-OH
3’-OH -C-T-T-A-A-G- 5’-PO4 5’-PO4 –G A-A-T-T-C 3’-OH 3’-OH -C-T-T-A-A G 5’-PO4 |
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What are the 4 steps involved in gene cloning?
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1. Isolate or synthesize the gene of interest
2. Incorporation of the gene into a cloning vector 3. Insertion of this vector into the host 4. Detection of the cloned gene |
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Name the 4 main methods of obtaining the target gene after gene cloning.
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-isolating directly from genomic DNA
-synthesized from an mRNA template via reverse transcriptase. -constructed from nucleotides in vitro. -Lyse the cell, then purify the DNA. (Then make a library of fragments and cut up the chromosomes and store them.) |
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Describe how to isolate the target gene through isolation from genomic DNA.
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1. Randomly with cleavage using various restriction endonucleases, cloning and selecting.
2. Isolating the gene fragment directly from an agarose gel, that is restricted, stained and removed from the gel. |
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Describe how to isolate the target gene through gene synthesized from mRNA template (4 steps).
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1. Using reverse transcriptase, it synthesizes single stranded DNA chains from RNA templates, complementary DNA (cDNA).
2. cDNA is made from each mRNA molecule. 3. The cDNA is then replicated by DNA polymerase to form dsDNA. 4. The dsDNA is inserted into cloning vectors, resulting in cDNA clones. |
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Describe how to isolate the target gene through synthesis from nucleotides in vitro.
There are TWO METHODS within this method. |
METHOD 1
1. If the nucleotide sequence of the target gene is known, the DNA could be synthesizedon a “gene machine”, or DNA synthesizer. 2. Short nucleotides synthesized of 20 to 30 oligonucleotides. 3. Stepwise addition of nucleotides to the 5’ growing end. Purified. METHOD 2 1. In Site directed mutagenesis, a short oligonucleotide is annealed to a single stranded copy of the gene of interest. 2. This is extended using DNA polymerase to produce a new copy of the gene with a mutated sequence. 3. This can be inserted into a host cell and studied. 4. Usually involves bacteriophage M13 since it is single stranded. |
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What is the genetic vehicle for insertion into a cell?
These vehicles have one or more BLANK BLANK (RE) sites that occur only once within the vector. These unique sites, without disrupting other areas of the vector, make it possible to... |
Cloning vectors
restriction endonuclease cleave the vector at a specific location |
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In the process of creating cloning vectors, digestion occurs with the restriction endonuclease, then the...
What is a method of using electrical current and rescue broth to get bacteria to take up DNA/cloning vector? This becomes a BLANK molecule. |
vector and gene of interest are mixed and ligated, and annealed.
Electroporation Recombinant |
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Cloning vectors have an origin called a BLANK.
Cloning vectors confer a detectable marker, BLANK marker, to the host cell The BLANK marker is often a BLANK resistance marker, which is incorporated into the antibiotic containing medium. Name two antibiotic examples. |
Ori
Selective marker selectable; antimicrobial Ex: tetracycline or ampicillin. |
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Review the example of how to use cloning vectors to make gold!
(Don't memorize) |
1. Digest the chromosome and pBR322 (vector) with SAL1.
2. Let it reanneal with T4 Ligase overnight. 3. Take the recombinant molecule and transform it into E. coli 4. To select for the gold gene, use TSA agar supplemented with Tc^5mg and Ap^25mg 5. Look for transformations Tc5 and Ap^R and look for gold |
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--Plasmids as cloning vectors--
Plasmids as CVs are small. How many bps in length? Consist of single or double stranded DNA? Have an origin of replication which allows... Maintained in multiple numbers within the cell so that... |
1,000 to 300,000 bp in length.
dsDNA them to replicate independently of the bacterial genome. amplification is possible. |
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--Plasmids as cloning vectors--
Have restriction sites that occur... Have selectable markers such as... Must be able to allow for... |
only once within the vector.
antimicrobial resistance insertion of foreign DNA. |
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What is a famous plasmid cloning vector?
How many copies are in your typical E. coli cell? This can be amplified to how many copies? What are the 5 sites on this plasmid cloning vector? |
pBR322
50; 2,000 EcoRI, HindIII, BamHI, PstI and SalI. |
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Which sites on the pBR322 plasmid cloning vector are within the Tc gene region?
Which sites are within the ampicillin resistance gene? Cloning into these sites results in BLANK BLANK and the resulting hybrid plasmids are referred to as BLANK. |
Tc: HindIII, BamHI, and SalI
Ap: PstI and EcoRI insertional inactivation; chimera |
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What bacteriophage is used as a cloning vector?
The wild type λ has BLANK# EcoRI sites and the modified phage has a BLANK EcoRI site. BLANK phages, another class of modified λ phage, contains deletions in their genomes that... |
The bacteriophage lambda (λ)
5; single Charon allow for the cloning of larger DNA fragments. |
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What is a cloning vector that moves DNA between say, G- E.coli and G+ Bacillus, and will replicate in both of them?
Name 3 examples from fastest to slowest. |
Shuttle vectors
Ex: E. coli (fastest), Bacillus (secretes products like B serine proteases), Psuedomonas |
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What are cloning vectors that contain regulatory sequences that allow for over expression of genes?
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Expression vectors
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What is a collection of cloned DNA fragments from the genome of an organism called?
What is a collection constructed from mRNA called? What is shotgun cloning? |
DNA library
cDNA library enzymatically cleaving entire donor genome into small fragments, screening (detection), the library is difficult. |
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What are the 3 usual hosts for cloned genes?
What are 3 methods of insertion of the cloned DNA into the host? Strong promoters on the vector will lead to... |
E.coli, Bacillus subtilis, Saccharomyces cerevisiae.
transformation, transfection, electroporation. high levels of gene expression. |
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What are 3 ways to detect cloned genes?
Briefly summarize/understand how autoradiography works |
Using protein products, antibodies, or autoradiography
--Autoradiography-- Use nucleic acid probes to recognize a specific DNA or RNA sequence of the target gene. Replicate colonies onto nitrocellulose filter and a reference plate. The colonies on the filter are lysed and their DNA is denatured by heating. A radiolabeled probe is added to the filter and is allowed to hybridize with ssDNA derived from the colonies. The probe will bind to complementary sequences. The colonies that hybridize are detected on x-ray sheets (autoradiography). |
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Who discovered Polymerase Chain Reaction in 1983? What is it used for?
What are the 3 steps of PCR? |
Kary Mullis; This technique is used to amplify DNA fragments in vitro.
1. The original dsDNA molecule is denatured at high temperature. 2. Oligonucleotide primers are annealed to the DNA at low temperature. 3. The primers are extended on the DNA template by a DNA polymerase. |
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--Detailed steps of PCR--
The steps are completed in a thermal cycle. As the two strands of the target DNA separate by heat denaturation... DNA polymerase is added and the DNA/Oligonucleotide primers are extended onto the DNA templates. After one round of this cycle... At the end of the second cycle, there are BLANK# copies. After 30 cycles, you have up to BLANK increase in copies. Many improvements in this cycle have been made. |
two synthetic oligonucleotide primers are added in excess.
there are two copies of the original DNA molecule. 4; 10^9 |
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There are several applications of rDNA technology. Name some, but there are six.
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rDNA technology has allowed for improved vaccines (subunit vaccines) that are more specific to target regions, allowing for more specific immune response.
Better detection of pathogens in the clinical laboratory through the use of specific nucleic acid probe development. Unique sequences used for the detection of the pathogen. Manufacture of recombinant proteins such as Humalin, a recombinant insulin molecule, and Interferon, which inhibits viral replication. Plant cells engineered with the Ti plasmid produced by Agrobacterium tumefaciens. This plasmid causes a tumor to form in a plant cell, acts as a shuttle vector between bacteria and plants. --Crown Gall disease (tumor forming disease). Ti plasmid replicates in plants. Protection of plant cells through the use of ice- mutants of Pseudomonas syringe. --This microbe/method is used to make artificial ice and for frost protection. Insertion of the Bt toxin genes into plant cells to make them produce the Bt toxin and thus kill insects that attack the plant. |
