Chapter 4 Flashcards
What does the term “electrophoresis” mean?
to carry with electricity
How do you run a gel?
- DNA fragments are loaded into an agarose gel
- gel is placed into a chamber filled with a conductive buffer solution
- a direct current is passed through
How does DNA pass through the gel?
since DNA fragments are negatively charged, they are drawn towards the positive pole, or electrode
What is the relationship between the rate at which a DNA fragment migrates and the size of its base pairs?
inversely related
Which travel further: smaller or larger DNA fragments?
smaller
What does TAE stand for?
Tris/acetic acid/EDTA
At what temperature does TAE need to be cooled before pouring?
55 C
What are other names for DNA size standards?
standards, ladders, rulers, or markers
How were the original DNA standards made?
made from bacteriophage cut with specific restriction enzymes that yielded DNA fragments of known number and size
Why is lambda phage DNA useful?
it covers a broad range of DNA sizes when cut with HindIII enzyme
Can you find the amount of DNA in a band if you have the intensity or thickness of the band? How?
yes, they are proportional
compare the intensity of the band to the intensity of bands in the DNA control to estimate the quantity of DNA
What are molecular mass rulers?
standards that are engineered to prod bands of evenly distributed molecular mass
What are other names for horizontal gel electrophoresis chambers?
gel tanks, gel boxes
what does molecular biology refer to?
it is the study of biology and the molecular level, also refers to a set of tools and techniques that have revolutionized biology.
what is the central dogma of molecular biology?
refers the the normal flow of genetic information from DNA to RNA and then to protein.
What two studies does molecular biology bridge between?
genetics and biochemistry
What are some molecular biology techniques?
restriction enzyme digestion analysis, ligation, bacterial transformation, and the polymerase chain reaction
What are some of the ways that microbiology has benefitted society?
scientists used molecular biology to trace the anthrax poisonings of 2001, pharmaceutical industries (Genentech and Amgen), exonerate wrongly accused criminals
What are horizontal gel electrophoresis chambers made from?
acrylic plastic; they have a positive electrode at one end and a negative electrode (cathode) at the other
What is sample loading buffer mixed with?
an agent such as glycerol or FicoII to increase the density of the sample so that it sinks into the well, and a dye to monitor migration of the samples
How long and at what voltage do gels typically run at?
100V for 30mins to 1 hr
What are restriction enzymes used for?
used extensively in recombinant DNA technology: involves moving DNA fragments from one location to another
What are restriction fragment length polymorphisms?
genetic differences/genetic variations among individuals, including mutations in restriction sites such that the sites are no longer recognized by the restriction enzyme
it generates a banding pattern unique for each individual
How does one perform RFLP analysis?
the DNA is digested with a restriction enzyme and the fragments generated are analyzed by agarose gel electrophoresis
What is Southern blotting?
genomic DNA is resolved on an agarose gel, denatured so that is single-stranded, and transferred onto a solid membrane of nylon or nitrocellulose
DNA bands are trapped inside the membrane and form a replica of the original gel
a probe is incubated in the membrane and is modified with a radioactive or fluorescent label so that it can be detected
the probe anneals to the single-stranded target DNA
the location of the site is visualized using fluorescence technology
who invented the central dogma of molecular biology?
Francis Crick
What happens in DNA replication?
an enzyme called DNA polymerase copies both DNA strands and makes a brand new copies of the DNA
What happens in DNA transcription?
enzyme RNA polymerase copies one DNA strand into messenger RNA, reads 3 prime to 5 prime
What happens in DNA translation?
the seqquence of bases on the MRNA is read and the information is converted into a polypeptide chain composed of amino acids, which folds into a protein
Special circumstances in which genetic information flows differently!
For ex, RNA viruses such as the influenza virus can replicate their own DNA. HIV can copy RNA into DNA, this is known as a retrovirus that uses reverse transcriptase.
What are DNA strands comprised of?
polymers of nucleotides, consisting of a sugar, a phosphate, and a nitrogenous base
What are the four nitrogenous bases?
Thymine, Guanine, Cytosine, and Adenine
Guanine and Adenine are…
Purines, two nitrogen bases
Thymine and cytosine are…
pyrimidines, one nitrogen base
A-T and G-C are
base pairs
what is the basis of PCR and DNA sequencing
complementarity
How many carbons does the sugar part of the DNA strand have? What does this mean it is called?
5; pentose
how are nucleic acids synthesized?
synthesized in vivid only in a 5’ to 3’ direction
what do the terms sense and antisense mean?
sense means 5’ to 3’, and antisense means 3’ and 5’
when DNA is transcribed , the antisense strand…
serves as the template for the synthesis of mRNA.
recombinant DNA technology…
based on the premise that dNA is virtually the same in all cells and can be acted on in other cells. the foundation of modern biotechnology.
example of genetic engineering
cotton plants, expresses a bacteria gene that makes them resistant to agricultural pests, in the pharmaceutical industry, recombinant proteins are produced in bacteria and eukaryotic cells.
restrictions enymes serve what purpose?
able to precisely cut DNA and enabled scientists to move genes from the genome of one organism into another genome of another, discovered by Werner Arber and Hamilton Smith and Dan Nathans
bacteriophages
invading viruses that inject their DNA, stopped by restriction enzymes, methyl groups prevent own DNA from being cut
enzyme name is derived from
bacterial genus, species, and then strain, name uses the first initial of the genus and the first two letters of the species name followed by the order in which the enzymes were discovered
restriction enzymes are more accurately known as
restriction endonucleases (endo means within, nuclease means an enzyme that cutes nucleic acids)
how is the DNA broken?
restriction enzymes cut the DNA at certain areas, stop sliding when they reach a certain combo, enzymes hydrolyze the sugar-phosphate bond, thus breaking the strand
blunt vs sticky ends
blunt-no unpaired ends, sticky-staggered cutes in the DNA, restriction sites are palindromic
separation of DNA with restriction enzymes can be separated using what
electrophoresis in an agarose gel
what is the function of ligase?
joins together one new cut piece of DNA to another genome by reforming the phosphate bonds that were broken by the restriction enzymes.
where is ligase seen in nature?
used in DNA replication to ligate the Okazaki fragments made on the lagging strand of DNA
What is more efficient, sticky ends with ligase or blunt end?
sticky end
What is likely to happen when putting a ligase into a plasmid?
re-circularizing is more likely, mostly because open ends of the plasmid chicken near each other
what happens to prevent plasmids from self-ligating?
the phosphate on the 5’ end of the plasmid is removed using an enzyme known as phosphatase in a process called dephosphorylation
dephosphorylation prevents what type of bond from forming?
phophdiester bond, can only circulate if fragment is inserted
what was the original supplier of enzymes for genetic engineering?
New England BioLabs
how is enzyme concentration denoted?
units of activity (U) rather than the concentration of the protein, definition is provided by the supplier, also supplied with a reaction buffer
unit of restriction enzyme
the amount of enzyme need to digest 1 microgram of lamda phase in 1 hour at 37 degrees
enzymes should be stored at, other conditions
-20 degrees celcius, do not heat/cool repeatedly, if working with them directly keep at -4 celsius
