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41 Cards in this Set

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What are restriction enzymes?
Enzymes which recognize and ind specific sequences in DNA: Recognition sites.
What are the types of restriction enzymes. What do they do?
1,2,3. Type 1 and 3 cleave a defined distance from the recognition sites. Type 2 cleaves DNA at and around the site.
What type of ends to restriction enzymes create?
Sticky ends
How does genetic cloning work?
Foreign DNA becomes combined with or inserted into a plasmid vector. Then the new plasmid is a Recombo DNA molecule which replicates and maintains foreign DNA fragments.
What is a vector?
A carrier of foreign DNA
What is reverse transcriptase?
Enzyme which synthesizes double stranded DNA from RNA template and is used to construct complementary DNA. If you don’t use a template, you use mixed RNA and the amount of cDNA produced is directly proportional to each RNA
What is gel electrophoresis?
A technique used to separate molecules based on their charge and molecular weight.
What are the two gels that are used?

Agarose and acrylamide.

What is the charge of DNA?
Negative
Towards what magnetic pole will DNA migrate in Gel electrophoresis?
Positive pole
How do you get bands in the gels in different spots?
The bigger the fragment, the slower the motion. The further down a fragment is, the small it is.
How do you tell if an organism has a particular gene in it’s genome?
Souther blot
How does a southern block work How has it changed with time?
You run a Gel electrophoresis. Then you coat it on the weird sponge and the weight and the thing to blot it with. Then you add a radioactive probe to the mixture and overlay the filter with XRAY film. Develop the film. Whatever bands appear on the film have that specific gene. Now we use fluorescents, and not radioactive stuff.
What technique did we learn that enables DNA amplification?
PCR
Where does the specificity of PCR arise from?
DNA primers and oglionucleotides.
What does the reaction mix of PCR contain?
Primers, Target DNA (template), thermostable DNA polymerase, dNTPs.
What is the cylcle for PCR?
DNA denaturation, Primers annealing to target DNA, Target DNA is synthesized, and repeat 34 times.
What are the problems with PCR?
You need to know the sequence of the DNA you’re amplifying.
What are the four types of cloning vectors?
Plasmids, phages/viruses, cosmids, and artificial chromosomes.
Why are plasmids good cloning vectors?
They replicate autonomously and are easy to purify.
What are the requirements for vectors?
An origin of replication, a selectable marker, and multicloning site/polylinker
When is a genomic library constructed?
When a gene of interest is on a chromosome that hasn’t been sequenced.
What are oglionucleotides
Short DNA molecules that flank the DNA sequence being amplified. (They serve as primers for DNA poly)
How do you construct genomic library
you cleave genomic DNA w/ restriction enzymes and clone each fragment into vectors. Every gene on the genome is cloned. Those plasmids are transformed into bacterial cells and the libraries are screened for the genes of interest in a variety of ways.
What are the most common hosts for recombinant DNA insertion?
E.coli and S. cerevisiae (eukaryotic host)
What do host cells lack (in terms of recombinant DNA insertions)?
Restriction enzymes and recA, they don’t have the ability to cut up the recombinant DNA
How do introduce DNA into microbes?
You do a transformation where you artificially induce competence with salts, or you electrocute it.
What is it called when you artificially induce competence with electricity?
Electroporation
Why would you want to express a foreign gene in a host cell?
Determine function and purify protein.
How would you express a foreign gene?
Expression vectors
What are expression vectors and what do they do?
They are vectors which contain inducible promoters which results in high level transcription with a selectable tag to help isolate the gene product.
How can a protein be isolated and purified via expression vectors?
A selectable tag
What is genomics?
The study of genomes
What is sanger sequencing and how does it work?
It was the most common method of determining DNA sequences. It works via terminating bases ddNTP’s. They will terminate synthesis and extend the DNA strand. You make the dna in pieces and eventually you will get the full DNA strand
What is the modern method of sanger sequencing?
You use 4 different fluorescent color dies instead of radiolabeled ddNTP. Electorphoresis and laser beam determines the order of the DNA sequence
What is NGS
Next generation DNA sequencing
How many strands can NGS sequence simultaneously?
10-100 billion bp/reaction
How many strands can sanger sequence simultaneously?
1,000 bp/reaction
What is shotgun sequencing?
A process to figure the seuqncing the genome sequence
What are the steps to shotgun sequencing?
Library construction to generate clones of portions of genomes. Random sequencing to determine sequences of clones. Fragment alignment and gap closure. Editing.
What is bioinformatics and why is it awesome?
It is a next gen sequencing that can do up to 640 DNA strands, or 10-100billion bp/reaction. It is the use of computers to analyze biological data…particularaly genomic and protomic data.