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E1 LEC 5 Molecular Genetics
Terms in this set (39)
what advantage does DNA have over RNA (proteins with amino acids)?
it has a lot of the same properties throughout the whole structure while RNA has many different amino acids with different properties
restriction enzymes are commonly found in prokaryotic /eukaryotic bacteria?
enzymes that recognize specific base sequences in FOREIGN DNA and cut these-- (i.e. an invading virus)
--host prokaryotic DNA is protected and remains in tact because the cleavage sites are protected by methylation
how is HOST PROKARYOTIC DNA protected from restriction enzymes that cleave foreign DNA?
they possess methylation sites at the places that would be cleaved
restriction enzymes recognize a specific _____ sequence meaning it is read in the same way way either direction (symmetrical), and then cut it out creating a double stranded break in the DNA
fragments of DNA cleaved by restriction enzymes that can be separated by gel electrophoresis and
once DNA is cut up by restriction enzymes and separated onto a gel does it need to be coated by SDS?
NO-- a nucleic acid does not need a coat because it already has a uniform negative charge; you can see the DNA on the fuel by staining it with fluorescent dye
-____ blotting: detects specific DNA fragments
-____blotting: detects specific RNA fragments
-_____ blotting: detects specific proteins using antibodies
-southern blotting: DNA
-northern blotting: RNA
-western blotting: proteins using antibodies
analytical method in which a complementary DNA strand (reverse complement AKA probe) has been radio labeled; a mixture of restriction fragments are separated by gel electrophoresis and blotted onto a nitrocellulose membrane; the DNA probe is added and hybridizes with the complementary DNA fragments-- the detection of the label reveals the position of the fragment of interest
southern blotting (used for DNA )
DNA sequencing (Sanger method):
-Four different reaction mixtures used containing DNA fragments that each have a _____ analog (ddATP, ddCTP, ddTTP, ddGTP) -- they are separated and placed on a gel electrophoresis:
1. a _____ is obtained from restriction enzyme digest and is recognized by DNA ____ to start the rxn
2. Each time a ____, ____ ____ analog is added into the primer chain there is a hard _____ in replication b/c the analog lacks the 3'-_____ group
1. primer, DNA polymerase
2. 2',3'-dideoxy analog, 3'-hydroxyl group
what method of DNA sequencing involves a hard STOP in the addition of more dideoxy analog bases by DNA polymerase, which STOPS replication process at those specific insertions?
what are the two reasons that the Sanger method for DNA sequencing works well?
1. the dideoxy analog incorporated into the primer chain creates a hard stop in the DNA sequence
2. the DNA can be separated by a SINGLE nucleotide
what method is used to AMPLIFY DNA sequences and is mainly dependent on altering temperature of DNA?
Polymerase chain reaction (PCR)
PCR (DNA sequencing method--amplifyinf ):
1. strand separation through ______
2. ______ (annealing): solution is cooled allowing each primer to hybridize to a DNA strand (preventing parent DNA duplex from reforming)
3.____ ____(extension of DNA): solution is reheated to optimal temperature for Taq DNA polymerase to synthesize copies of both strands
3. DNA synthesis
advantages of PCR DNA amplification method?
-PCR can be amplified for many cycles
-very small amounts of starting material can generate huge amounts of product
-basis for many diagnostic tests
What are the two key tools in recombinant DNA?
DNA ligase and restriction enzymes
what method of DNA sequencing is useful for putting multiple genes/domains together if splicing is not working?
when genes are cloned what is basically happening to them?
they are amplified many times
Genes can be cloned by inserting them into cell ____ where they replicate as cells divide; this is achieved by attaching a DNA fragment into a vector that is capable of replicating autonomously in the appropriate host
Recombinant DNA process:
1. _____ enzymes cleave DNA at specific sites and produces complementary single strand with "____ ends"; any other DNA fragment can then be inserted into that splice site if it has those same ends
2. The DNA fragment and cut vector are joined together by DNA _____ (catalyzes a phosphodiester bond at a break in the DNA chain)
1. restriction enzymes
2. DNA ligase
what can be used to clone A LOT of bacterial DNA?
plasmids or vectors -- able to replicate within their own circular duplex and they possess lots of restriction sites for DNA to insert into
vectors can be used for what two things?
1. cloning and screening DNA
2. amplification of DNA
plasmids have multiple ____ sites in the _____ region where foreign DNA can be spliced in using restriction enzymes and DNA ____
what are the 3 key features of a plasmid?
1. polylinker region
2. selection mechanism (positive selection)
3. origin of replication
Ex Plasmid: _____: carries genes for resistance to B-lactan (ampicilin) and a B-gal enzyme to kill only what is neccessary and save the plasmid
complementary DNA (cDNA) is made from mRNA using an enzyme called what?
Recombinant DNA is complementary in to mRNA rather than genomic DNA--> this allows human genes to be expressed in _____
getting cDNA from mRNA:
1. viruses use _____ _____ (enzyme) to form a DNA-RNA hybrid to replicate their own genome (DNA is complementary to RNA)
2. RNA-cDNA is then hydrolyzed by raising the ____ (DNA is more resistant to -OH than RNA is)
3. The complementary strand is synthesized be adding a new ____-- synthetic ends can be added and cDNA is inserted into a vector to be cloned
1. restriction enyzmes
enzyme that removes bases from the ends of nucleic acids to make a modification in the protein's sequence
engineering a PCR primer w/ a mutation you want to make and amplifies the mutated piece of the DNA
viral transduction of genes allows for the modification of gene expression in what?
a variety of tissues
viral transduction of genes allows for modification of gene expression in a variety of tissues;
-the ____ of the virus (viral coat protein) determines the target tissue
-the ____: directs cell type specific expression
method used to REDUCE the expression of a target gene
RNA interference (RISC complex and siRNAs)
reducing the expression of a target gene:
1. ___RNA is recognized by eukaryotic cells as "foreign" inducing a degenerative response
2. ____ enzyme cleaves the dsRNA into 21 siRNAs
3. strands are separated and bind to a _____ complex
4. The complex cleaves the mRNA complementary to the bound fragment
3. RNA induced silencing complex (RISC)
method (reducing expression or knocking gene out) that involves homologous recombination (crossing-over) of a mutated gene into your target gene to reduce the expression of the gene
target dene disruption
method of gene disruption that allows you to knock out a specific target gene in a specific tissue
Cre Lox system
Cre Lox gene disruption method:
-used to knock out a specific target gene in a specific __
-relies on making two transgenic mouse strains:
1) strand one: has a ____ recognition site flanking the region to be deleted
2) strand two: contains Cre ____ gene downstream of the tissue specific promotor that controls the recombinase expression
-LoxP recognition site
-Cre recombination gene
what method is used to protect bacteria from viruses using the cas9 enzyme?
CRISPR Cas9 Gene editing
CRISPR gene editing uses the ____ enzyme to cleave specific regions of DNA and then refill those gaps with something that will knock the gene out and protect the bacteria from foreign DNA virus
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