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32 Cards in this Set

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What is recombinant DNA?

What requirements are needed to make recombinant DNA?
"artificial" or "manufactured" DNA

Need:
1) Restriction endonucleases--cuts DNA
2) Vectors--carries fragments of DNA into host cell.
3) Source of DNA
What are restriction endonucleases?
Cleave DNA at specific DNA sequences
What are the two types of restriction endonucleases?
"sticky ends"--endonuclease cleaves double stranded DNA-->makes 4, different DNA pieces. Strands have "overhangs" that are complementary, so will get back together when mixed together-->BAM HI

"blunt ends"--endonuclease cleaves double-stranded DNA-->makes 4, different strands-->have blunt ends-->will not get back together when mixed together--HAE III
What are types of vectors that can be used?
1) Plasmid--circular DNA that replicates independently of host cell DNA--confers antibiotic resistance to host cell.
2) Phages--linear DNA--uses bacteria as hosts.
3) Viruses--contain DNA--use mammalian cells as hosts.
What are the sources of DNA?
1) Genomic DNA-uses restriction enzyme to digest chromosomal DNA into tiny pieces (shot-gun genomic cloning)

2) DNA made by chemical synthesis

3) mRNA--DNA made by reverse transcriptase. (mRNA-->mRNA/DNA-->cDNA-->double stranded DNA

cDNA--no introns
What is a palidromic DNA sequence?
--Sequence of DNA that reads the same in both (5-->3 and 3-->5) directions.
--Lots of restriction endonucleases recognize these sequences and cut them.
What are major steps in construction of recombinant DNA molecule into a plasmid?
Restriction endonuclease cleaves parent DNA-->DNA containing blunt or sticky ends is inserted into the plasmid as a vector-->DNA is transformed into bacteria.
What are major steps in reverse transcriptase?
mRNA template with oligo dT primer-->reverse transcriptase/dNTP's bind to primer-->elongate-->get mRNA-DNA hybrid-->mRNA removed by alkaline digestion-->get cDNA-->reacts with DNA polymerase I, dNTP's-->get double-stranded DNA!
What is major difference between cDNA clones (made by reverse transcriptase) and DNA clones (made from genomic DNA)?
Genomic cloning: DNA-->restriction nuclease digestion-->DNA fragments-->DNA cloning-->genomic DNA clones

cDNA cloning: DNA-->transcription-->RNA transcript-->RNA splicing-->mature RNA-->reverse transcription-->cDNA

genomic DNA--contains exons AND introns-->introns present, so no functional protein, but can be used as diagnostic tool.

cDNA cloning-->introns are REMOVED by splicing-->can make a protein.
What is overall schematic for making a clone that carries gene of interest?
Eukaryotic DNA put into bacterial plasmid-->E.Coli added to plasmid-->grown on plate-->bacteria lysed-->exposes protein that was added-->transfer protein to nitrocellulose sheet-->probe w/antibody-->tells you if gene is being expressed!
Why is cloned DNA useful?
-Templates make human proteins for treatment
-Molecular probes can be used for clinical diagnosis
-Source of normal gene for gene replacement therapy
What are the basics of nucleic acid hybridization?
-->Process by which the single-stranded probe unites with complementary DNA in an unknown sample.

Two DNA strands will hybridize if they are perfectly complementary. If not, a kink will form which will destabilize the DNA molecule.

Used in: Southern blotting (DNA-DNA hybridization) and Northern blotting (DNA-RNA hybridization)
What are basics of Southern blotting? What is it useful?
-->DNA-DNA hybridization

DNA isolated-->digested with restriction endonucleases (#fragments=#recognition sequences)->fragments separated by electrophoresis (smaller fragments move faster)-->DNA denatured and transferred to nitrocellulose paper-->gives replica of pattern on cell-->paper treated with radioactive/flourescent DNA probe-->will hybridize to DNA fragment w/ complementary nucleotide sequence-->radioactive probe produces dark image on x-ray-->flouresent probe will make flourescent spot detected by detector

*DNA PROBE HYBRIDIZES WITH DNA ON NITROCELLULOSE BLOT OF A GEL

Used to--
-Determine if gene is present in tissue of interest
-Determine copy number of gene
-Determine gross alterations in a gene.
What are basics of Northern blotting? Why is it useful?
--Similar to southern blot, but RNA species is separated/transferred (instead of DNA)-->DNA-RNA hybrid will be detected the same way as in Southern blotting

Used to--
-Determine whether gene is expressed as mRNA
-Determine structural alteration in mRNA molecule.

*DNA PROBE HYBRIDIZES WITH RNA ON NITROCELLULOSE BLOT OF A GEL
What are basics of Western blotting? Why is it useful?
Proteins separated by gel electrophoresis-->probed with ANTIBIOTICS that bind a specific protein

--If protein is present, will flouresce with with flourescent probe
--If not, you'll see nothing.
What are basics of DNA microarray analysis?
Have normal and abnormal cells-->mRNA-->reverse transcriptase created cDNA-->label cDNA with flourescent molecules (red--cancer, green--normal)-->mix cDNA's and put on microarray-->microarray has wells with sequences for different genes-->cDNA hybridizes to the single stranded DNA already on the chip-->hybridization produces a color on the chip that is different from the color of non-hybridized DNA.


if spot is RED on microarray--mutated cell produces more of its message
if spot is GREEN--normal cell produces more of its message
if spot is YELLOW--both cells produce message equally
if spot is BLACK--neither cell produces message

DIFFERENT than southern/northern blot-->in those, you know what gene you're looking for--in microarray, looking for differences in genes
What are basics of Polymerase Chain Reaction (PCR)?
-->makes TONS of copies of a DNA sequence.

DNA heated-->separates into individual strands-->single strands bind primers which are complementary to sequences on opposite strands and define sequence to be amplified-->DNA polymerase extends primers-->synthesizes 2 DNA strands complementary to original DNA molecule-->repeated several times-->amplified product!
What are DNA probes?
--single strand of DNA that can hybridize (base pair) with complementary sequence on another single-stranded polynucleotide of DNA or RNA
--must contain a LABEL
--commonly labeled w/radioactive or non-radioactive agents by 5'-end labeling, random primer labeling, or nick translation.
Describe use of recombinant DNA technology in prenatal diagnosis
--Can detect severe genetic defects during pregnancy.

-->Can use restriction mapping and Southern blots to detect point mutations (sickle cell anemia)--Southern blot will show ONE, big fragment, because it is not cleaved by restriction fragments, because of mutation.
-->Can use Southern blots to detect deletion, insertion, or rearrangement (HGPRT, thalessemia)--mutant will have bigger fragment, because mutated DNA is not cleaved correctly.
-->can use PCR w/nested primers to test for pre-implantation diagnosis of most common form of CF--caused by three-based deletion of gene.
Describe use of recombinant DNA technology to identify infectious agents
PCR can amplify small amounts of DNA-->allows detection before clinical symptoms appear or prior to presence of Abs directed toward virus.
What is promotor blocking?
-->type of GENE THERAPY

provides target cell with fake/decoy promotor-->specific TF's directed away from real promotors-->stops transcription!

blocks TRANSCRIPTION
What are antisense nucleic acids used for?
-->type of GENE THERAPY

complementary oligonucleotides to known sequence of mRNA can be used to inhibit translation of mRNAs.

blocks TRANSLATION

-->antisense RNA hybridizes w/sense RNA-->blocks translation of normal mRNA

--has been used in trials to inhibit: oncogenes (CML), or viral diseases (AIDS)
What is gene replacement therapy?
Introduce gene to replace a defect!

Ways to introduce DNA:
1) Electrophoration-->apply ELECTRIC FIELD--"shock" DNA into cell-->NON-SPECIFIC
2) LIPOSOME-->put into immunogenic lipid-->NON-SPECIFIC
3) RECEPTOR MEDIATED ENDOCTOSIS-->link DNA to ligand of specific receptor-->gets into cell-->MORE SPECIFIC--need correct receptor receptor
4) VIRAL VECTORS-->adenovirus, retrovirus, or herpes simplex virus-->"TRANSFECTION"-->introduced into site where gene is needed!
Describe basic process of viral vector introduction into cell (example seen in SCID tx)
-->use viral vectors to deliver DNA into cells.
-->viral vectors are modified to remove harmful genes (gag, pol, env)
->can add gene (for example: ADA) to "carrier" virus-->introduce into cell
->can be used for in vivo delivery of exogenous DNA
What are disadvantages to using viral vector introduction?
--Viral DNA is synthesized by reverse transcriptase and DNA polymerase is inserted randomly.
--If inserted into critical site, could have major consequences for target cell-->ie: "insertional mutagenesis" (exogenous gene activates oncogene or suppresses tumor suppressor gene--causes cancer)
What is ex vivo gene transfer?
--target cells removed from patient and genetically modified in lab-->transfected cells are selected and reintroduced into patient

-->clone normal gene into vector that will be incorporated into host DNA--introduced gene will be transcribed-->makes protein-->corrects deficiency in host
Describe ex vivo gene transfer
--want to clone normal gene into a vector that will be incorporated into host DNA
-->take DNA out of patient-->add genes in lab-->put back into patient
What is clinical example of ex vivo gene transfer?
-->SCID!
--SCID is inherited disorder caused by mutations-->decrease T, B cells, NK cells.
-->usually a defect in gamma-c gene or ADA.

To correct defect in gamma-c gene (SCID X-1):
-->bone marrow stem cells obtained, cultured, and transfected w/retroviral vector carrying NORMAL gamma-c gene-->injected back into patients-->after tx, both infants expressed the gamma-c genes

To correct ADA deficiency
Bone marrow cells obtained from pt-->transfected w/"ADA carrier" DNA-->transfected marrow cells recombined-->transplanted back into patient-->ADA is now present!
What are advantages/disadvantages to using adenovirus vectors?
retrovirus-->RNA virus--needs reverse transcriptase
adenovirus-->DNA virus--no reverse transcriptase needed

advantages
-->adenos infect a WIDE RANGE of cell types
-->adenos can transfer genes into non-dividing cells.
-->adenos are generally non-integrating--introduced gene products are expressed from episomal DNA

disadvantages
-->episomal adenos are often lost after cell division, making them unsuitable for long-term expression
-->most adenos are immunogenic
-->adeno vectors exhibit wide host-cell range, so may hinder in-vivo targeting to a specific cell type
Describe TH gene in astrocyte
Example of introduction of a gene of interest by expressing gene under control of promotor

-->expression of TH under control of GFAP promotor-->recominant DNA injected into rat model of parkinson's-->expression detected by PCR and TH protein by immunoassay
What is PCR with nested primers?
--Uses TWO primers to hybridize DNA--so, DNA is digested twice.
--allows you to amplify region that was already amplified by first round--makes a more detailed amplification.
--good for things like CF, because it's only 3 nucleotides, which is tiny!
why not just more rounds?
--DNA polymerase can make mistakes-->reduces amplifications to prevent mistakes.
What is ex vivo vs. in vivo?
Ex vivo--cells removed from host-->manipulated in lab-->put back into host

In vivo--vector injected directly into host