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Recombinant DNA Tech I
Terms in this set (60)
restriction enzymes and generation of DNA fragments contain...
defined terminal sequences
Restriction endonuclease recognition sites are short and usually...
palindromic DNA sequences - EcoR1 example as a protector or type of immune system
an enzyme that can cut the DNA into sticky ends
EcoR1, typical example
for a long stretch of DNA..
there are many DNA recognition sites for cutting
Once you cut the DNA with an endonuclease, what is the next step?
analysis of the fragments - agarose gel electrophoresis to test the mustures of restriction fragments
In an agarose gel, which will run faster? long or short DNA fragments
short fragments go through the gel pores quickly
How quickly can you run a PCR test these days?
in around 1 hr
How quickly can you run analysis of an entire human genome?
typically 1 week
In the old days, had to analyze a DNA fragment. know the size of the fragment and reach a conclusion. Run long gels, analyze every band. why?
in order to read the full sequence. now we don't have to do this fragment by fragment
Enzyme restriction sites are...
What sequencing is still being used in FBI labs?
Restriction Fragment Length Polymorphism (RFLP) analysis
RFLP can also be used to diagnose what disorder?
sickle cell anemia genotypes
What molecule does bacteria use to prevent digestion of their own DNA via EcoR1?
For sickle cell anemia, if the patient does NOT have a mutation, HP1 will be able to?
digest and cut the DNA at its specified spot. by the size of the fragment, you can quickly tell if the patient has this disease or not
If you are given 10 million base pair fragment and you digest with a single enzyme, you will see...
a smear in the blot, no clean blocks will appear
If you have this smear, then you will use what test?
a southern blot
How time consuming is southern blot?
2 days worth from start to finish, not safe because it is a biohazard for phosphorescence
look at wikipedia for southern blot, PCR, and endonuclease topics!!
If you want to detect DNA, the tech is called?
If you want to analyze RNA, the tech is called?
After we found out how to excise and fragment the DNA, what comes next in the the process?
amplification of the fragments for analysis - PCR (cloning into a vector)
the cloning of DNA began in 1976 with what accomplishment?
the creation of recombinant DNA molecules in vitro
the idea is that you have sticky ends and if you cut the DNA with the same enzyme you will have complementary ends. And when you mix these fragments what happens?
transformation and ligation of the ends
the idea behind creation of recombinant DNA molecules in vitro is to have?
identical restriction enzymes cutting the separate DNA strands
what amplifies the DNA after it is cloned?
usually E.coli bacteria
the reason for the mass DNA proliferation is the presence of what?
origin sites on the DNA plasmids
The plasmids can keep replicating themselves, specifically HOC genes can get up to what number in the bacterium cell?
around 100 copies of the same plasmid
what is a cloning vector from lecture - first one of its kind?
pBR322 - via this vector the labs at UM cloned 2 drug resistant genes into one plasmid. you can activate one drug resistant gene by inserting a foreign DNA fragment into a drug resistant gene
If you insert a foreign DNA fragment in a drug resistant gene then you disrupt the reading frame and introduce?
frame-shifting, possibly a stop codon and mess up that gene
If the plasmid has an AMP resistant gene and a TET gene, but the TET has an insertion then?
the cell still has resistance to ampicillin but not to tetracycline
What allows you to clone the DNA in PCR?
What is another way to clone the DNA?
phage llambda vector - allows you to insert a very long DNA fragment (20kb) compared to the smaller plasmid. used more in the old days
What is used to determine the number of infectious particles in a viral suspension?
a plaque assay
in the old days, people use phage llambda to construct what?
when ppl construct a DNA library, they chop the DNA into fragments and only purify DNA of certain lengths (20kb) and then what?
then ligate this DNA to llambda factor
the difference between partial and complete DNA digestion??
complete means all the restriction sites will be linearized by the enzyme and partial means only 20-30% are linearized
the chances are if you clone enough fragment..
you will cover the whole genome because some restriction enzyme sites will be intact
if you did a complete digestion of the human genome, then say a gene X could be segmented/cut and
you will not be able to clone this gene
if you do a partial digestion, then you will have larger fragments and can cover more intact genes than...
complete digestion and then replicate these fragments into a DNA library
the DNA can ligated to a what?
the vector can then be what?
amplified for analysis
some vectors are for DNA libraries, but sometimes you want to express the protein. what do you do?
you need a promotor recognized by the host (Ecoli and lac gene) DNA replication enzymes
human cell cannot recognize the promotor of bacteria genes - Y/N?
the promotor on the gene is recognized by?
RNA polymerase, then mRNA, then ribosome for protein synthesis
What type of gene promotes cell growth?
Cancer cells don't do what?
they don't age (MCS7 or Hiela cell) they grow without any control
they chop up the DNA from a ca patient, and the fibroblast will grow very fast. this is the process of how they isolated and cloned the first what?
you have foreign DNA transformed into the mouse fibroblast. because it is an oncogene it does what?
promotes cell grow and we can purify it - we always need a selective marker for the foreign DNA
A human oncogene transformed into a mouse cell can promote uncontrolled cell growth. Y/N
Construction of a cDNA library there are how many steps?
4 steps - don't have to memorize all steps but how the difference between this and genomic DNA library
in genomic DNA libraries, you chop up all of the DNA from the cell and clone everything into the library. Y/N?
With the cDNA library you clone the purified mRNA after splicing so only exons are left, and you then do what?
do purification of mRNA, reverse transcription, and clone to a specific vector - this is called a cDNA library
Once you have the library with many many colonies which can host different genes. what do you do at this point?
screen and separate the colonies
each colony will host?
for a DNA library, how many perti dishes?
Once you have the petri dish, you can kill the E.coli on the covering membrane sheath, some Ecoli will be attached. you kill these, denature, and release DNA and??
introduce the probe - southern blot probe
what does DNA denature mean?
separate the two sides - single stranded
Can you now synthesize a 100kb fragment?
no problem! labs will do this for you
Can you use antibodies to identify expressed clones?
absolutely! using it to pick up the target gene
Transcription will produce what?
pre mRNA, the pre mRNA will be spliced, splicing will remove introns, then you have mature mRNA which is the template for protein synthesis --> that's the cDNA library foundations
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