Flashcards in DNA Lecture 3 Deck (27):
The information for biological structure and
function is encoded in the ___________
sequence of the DNA molecules in cells.
What category structure is this referring to?
Naturally occurring DNA molecules are large or small?
Large about 4 million bases in each strand of E. coli
To sequence DNA, the first step is that it must be fragmented into manageable and defined pieces. What accomplishes this step?
The second step of DNA for sequencing is that we must obtain a significant amount of each fragment to work with. What accomplished this step?
Molecular cloning, or recombinant DNA technique
Defined pieces of a large DNA molecule can also be isolated by the biochemical technique of _____ which separates molecules by size
What were the two sequencing techniques developed that depend upon nucleotide chemistry, DNA metabolism, and electrophoretic methods
This DNA sequencing method's use is more widespread, technically easier.
This DNA sequencing technique uses two basic reaction to cleave a polynucleotide chain wherever a particular residue (A, T, G or C) is found
The Sanger method uses synthetic 2',3' - dideoxynucleoside triphosphates and DNA polymerase (enzyme involved in DNA replication)
In the ddtp structure, both of the 2' and 3' carbon atoms of ribose DO or DO NOT have an OH group
DO NOT have an OH group
When DNA is synthesized, one strand acts as a
template for the synthesis of the complementary strand
An enzyme called ________ catalyzes
the sequential addition of nucleotides to the 3’ end of the growing strand.
Chain elongation with the DNA polymerase enzyme requires two things.
A single unpaired strand (template)
A short primer strand to provide a a free ___________ group at the 3' end, to which a new nucleotide is added
Hydroxyl (OH) group
Each incoming nucleotide which is added to the primer strand is selected by base pairing to the appropriate nucleotide in the _____
In cells, the 3' hydroxyl group of the primer reacts with the incoming deoxynucleoside triphosphate (dNTP) to for a new
The fundamental reaction of DNA synthesis by DNA polymerases is a
Phosphoryl group transfer
What is the nucleophile during chain elongation?
Where does nucleophilic attack occur?
What is released in the reaction ?
The 3'hydroxyl group at the 3' end of the growing strand
The alpha phosphorous of the incoming deoxynucleoside 5' triphosphate
Inorganic pyrophosphate (2 phosphates?)
Hydrolysis of the pyrophosphate molecule in chain elongation provides some of the ___________ for the reaction
Thermodynamic driving force
The sanger procedure uses ________ triphosphate analogs to interrupt DNA synthesis
When ddNTP is inserted in place of a dNTP strand, strand elongation is halted after the analog is added to the primer strand. Why?
Because ddNTP lacks the 3' hydroxyl group needed for the next step
In the Sanger method, the unknown, single stranded DNA is mixed with an oligonucleotide complementary to the known sequence of the plasmid, which is directly next to the unknown segment.
The oligonucleotide binds to the known sequence through base pairing and acts as a ___ with a free 3' OH group for the synthesis of a new strand of DNA
In the Sanger method, four reaction solutions are prepared which contain the single stranded DNA with bound primer, a mixture of 2' deoxyribonucleoside triphosphates, an alpha-32 Phosphorous labeled 2'deoxyribonucleoside triphosphate, and DNA polymerase
The P radioisotope allows one to determine the ___ of the fragment on a gel by autoradiography
In the Sanger method, one of the four 2',3' dideoxnucleoside triphosphates is mixed into each tube, at a concentration 100 times lower than the dNTP concentration. Chain growth is terminated, and producing base specific labeled fragments. Why does growth terminate?
There is no 3' OH group for a subsequent nucleotide to react with
In the Sanger method, the length of each synthesized fragment is a measure of the number of nucleotides from the primer to the dideoxynucleotide
In the Sanger method, the four reaction solutions are added to a gel and subjected to _______ (PAGE or agarose)
It separates the synthesized fragments by ____
Exposure of photographic film to the gel allows
an identification of bands. Because the fragments are radio labeled at their 5' ends with 32 P, only the fragment to the 5' side of the break is visualized.
Because shorter DNA fragments migrate
faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the
Reading the gel from the bottom to the top
corresponds to the sequence from the 5’→ 3’ end of the non-template strand (the primer strand)
The sequence of the template strand is the ______ of the sequence read from the gel.
Typically, ______ bases in the sequence can be determined by the dideoxy method