DNA Lecture 2 Flashcards
It is the nucleic acid covalent structure and the nucleic acid sequence, and it stores genetic information
Primary Structure
What are three ways to represent pentadeoxyribonucleotide?
pA-C-G-T-A(OH)
pApCpGpTpA
pACGTA
a primary structure composed of less than or equal to 50 nucleotides is a(n)
oligonucleotide
a primary structure composed of more than 50 nucleotides is a(n)
polynucleotide
any regular, stable structure taken up by some or all of the nucleotides in a nucleic acid
Secondary Structure
Complex folding of large chromosomes within eukaryotic chromatin and bacterial nucleoids
Tertiary Structure
What did the experiments of Avery, MacLeod, and McCarty (1944) provide evidence of?
That DNA is the bearer of genetic information
How did Avery, MacLeod, and McCarty provide evidence that DNA is the bearer of genetic information?
DNA extracted from a virulent strain of bacterium was injected into a nonvirulent strain of the same bacterium, it genetically transformed the nonvirulent strain into the virulent strain
How did Hershey and Chase (1952) provide evidence that DNA carries genetic information?
they used radioactive phosphorous (32p) to tag the DNA and radioactive sulfur to tag the protein viral coat, and showed that when the virus infected its host cell (E. coli), the phosphorous containing DNA of the viral particle enters host cell and provides genetic information for viral replication
the sequences of bases from the 5’ end to the 3’ end in a nucleic acid
•e.g. pACGTA
Primary Structure
The sequence of nucleotide bases encodes the sequence of _______ in a protein
A sequence of three nucleotides provides a ____ for an amino acid
amino acids
codon
DNA of eukaryotes is in the
Protein synthesis occurs on ___ in the ____
what is the carrier molecule?
nucleus
ribosomes in the cytoplasm
messenger RNA (mRNA)
acts as an intermediary by using information encoded in DNA to specify the amino acid sequence of a functional protein
mRNA
The triplet codons in mRNA which specify a certain amino acid sequence are referred to as the
standard genetic code
T/F Most naturally occurring DNA molecules are too big to determine the sequence of the whole molecule in one operation.
True
How can the sequences of nucleotide bases (i.e. primary structure) in DNA be determined?
Chemical cleavage method (Maxam and Gilbert) Dideoxy method (or chain-terminator method developed by Sanger)
Both the chemical cleavage method and the dideoxy method of determining the sequences of nucleotide bases in DNA depend upon starting with a ________, many molecules corresponding to one segment of the extended DNA molecule
homogenous sample of DNA
In the first step of DNA cloning, DNA is cut at precise locations. The DNA segment is prepared by treating the isolated DNA with ______
An enzyme
The enzyme that is used to prepare DNA segments in cloning catalyzes hydrolytic cleavage within a nucleic acid chain, called an
endonuclease
These nucleases cleave DNA only at specific recognition sequences to generate a set of smaller fragments
For example, the enzyme EcoR1 recognizes the sequence 5’ GAATTC 3’ and hydrolyzes what bond between G and A?
Restriction endonucleases
The phosphodiester bond
How many types of restriction endonucleases are there?
3 (Type I, II, and III)
These restriction endonucleases are large and cleave the DNA a certain number of base pairs away from the recognition sequence and require energy of ATP
Type I and III
This type of restriction endonuclease is simpler than the others, requires no ATP, and cleave the DNA within the recognition sequence itself
Type II
Treatment of DNA with restriction endonucleases will result in a finite number of fragments, which correspond to __________ at each of the recognition sites
Hydrolysis
Do restriction endonucleases cleave both strands of DNA?
What are at the ends?
Yes.
Fragments with unpaired nucleotides
Is it possible to separate all of the fragments from one another to gain more purity after treatment with restriction enzymes?
No. Impossible.
In step 2 of DNA cloning, a known small molecule of DNA is selected, and it is typically a plasmid or viral DNA
Cloning Vector
A circular DNA molecules that replicates separately from the host molecule and is typically selected for as a cloning vector
Plasmid
What is a cloning vector (plasmid) treated with?
What does this achieve?
The same restriction enzyme that the DNA was treated with
The ends of the cleaved plasmid DNA matches those of the unknown fragments
In step 3, the DNA fragments are joined covalently. What links the cloning vector and the DNA to be cloned? It’s another ‘special’ enzyme
DNA ligase
What bonds are formed between the ends of the DNA fragments and the cleaved plasmid molecule?
What is the name of the structure that results?
Phosphodiester bonds
Recombinant DNA molecules
In step 4, recombinant DNA from the test tube is moved to the host cell where _______ can occur
DNA replication
When recombinant DNA is moved to the test tube suspension, the bacteria can take it up from the solution. How much DNA can a typical bacterium take up?
A single molecule
In step 5, the bacterial cell suspension is spread on an agar nutrient plate and allowed to grow. Colonies form which correspond to a single cell that has been multiplied.
T/F: Each cell contains one copy of one recombinant molecule
False. Each cell contains many replicated copies of one recombinant molecule. Molecules containing one fragment can then be isolated from a culture of a single colony.
Growing cells on an agar plate in the presence of an antibiotic allows for what?
Selections of colonies with and without the DNA
What are the most common vectors?
Why are they useful?
Plasmids and bacteriophage chromosomes
They replicate independently of the host cell
In our example of vectors, what E.coli plasmid is used as a vector? It contains a high origin of replication and genes conferring resistance to ampicillin and tetracycline (antiobiotics)
pBR322
To allow for further study, the recombinant DNA is separated from the plasmid. What can be used as a treatment to release the fragment from the plasmid?
The fragment can be separated from the plasmid by a technique called
The restriction enzyme used earlier (in our example, EcoRI)
Gel electrophoresis
What are the two types of gel electrophoresis?
Agarose and polyacrylamide
Type of gel electrophoresis that is a cross-linked polysaccharide
Type of gel electrophoresis that is a cross-linked polymer of acrylamide and N,N’-methylenebisacrylamide
When acrylamide is polymerized in water, what is formed?
Agarose
Polyacylamide
Gel
What cause the particles to migrate in gel electrophoresis?
An electric field, the particles are charged
What charge do DNA molecules have?
Therefore, they move towards the cathode or anode?
Negative
Towards the anode (anions move toward the anode)
In agarose and PAGE electrophoresis, the rate of movement of molecules depends upon what, due to the sieving effect of the gel?
Their size
Do larger or smaller molecules move more rapidly through electrophoresis?
The smaller/shorter molecules moved more rapidly and are found farthest down the gel.
