DNA Lecture 3

Question 1

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?

Answer 1

nucleotide base

Primary Structure

Question 2

Naturally occurring DNA molecules are large or small?

Answer 2

Large about 4 million bases in each strand of E. coli

Question 3

To sequence DNA, the first step is that it must be fragmented into manageable and defined pieces. What accomplishes this step?

Answer 3

Restriction endonucleases

Question 4

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?

Answer 4

Molecular cloning, or recombinant DNA technique

Question 5

Defined pieces of a large DNA molecule can also be isolated by the biochemical technique of _____ which separates molecules by size

Answer 5

Gel Electrophoresis

Question 6

What were the two sequencing techniques developed that depend upon nucleotide chemistry, DNA metabolism, and electrophoretic methods

Answer 6

Sanger

Maxim-Gilbert

Question 7

This DNA sequencing method’s use is more widespread, technically easier.

Answer 7

Sanger

Question 8

This DNA sequencing technique uses two basic reaction to cleave a polynucleotide chain wherever a particular residue (A, T, G or C) is found

Answer 8

Maxam-Gilbert

Question 9

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

Answer 9

DO NOT have an OH group

Question 10

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.

Answer 10

DNA polymerase

Question 11

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

Answer 11

Hydroxyl (OH) group

Question 12

Each incoming nucleotide which is added to the primer strand is selected by base pairing to the appropriate nucleotide in the _____

Answer 12

template strand

Question 13

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

Answer 13

Phosphodiester band

Phosphoryl group transfer

Question 14

What is the nucleophile during chain elongation?

Where does nucleophilic attack occur?

What is released in the reaction ?

Answer 14

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?)

Question 15

Hydrolysis of the pyrophosphate molecule in chain elongation provides some of the ___________ for the reaction

Answer 15

Thermodynamic driving force

Question 16

The sanger procedure uses ________ triphosphate analogs to interrupt DNA synthesis

Answer 16

Dideoxynucleoside

Question 17

When ddNTP is inserted in place of a dNTP strand, strand elongation is halted after the analog is added to the primer strand. Why?

Answer 17

Because ddNTP lacks the 3’ hydroxyl group needed for the next step

Question 18

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

Answer 18

Primer

Question 19

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

Answer 19

Position

Question 20

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?

Answer 20

There is no 3’ OH group for a subsequent nucleotide to react with

Question 21

In the Sanger method, the length of each synthesized fragment is a measure of the number of nucleotides from the primer to the dideoxynucleotide

Answer 21

!!!!

Question 22

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 ____

Answer 22

Electrophoresis

Length

Question 23

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.

Answer 23

!?

Question 24

Because shorter DNA fragments migrate

faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the

Answer 24

Primer (5’end)

Question 25

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.

Answer 25

complement

Question 26

Typically, ______ bases in the sequence can be determined by the dideoxy method

Answer 26

250

Question 27

DNA sequencing is readily automated. In a variation of Sanger’s method, the dideoxynucleotides used for each reaction are labeled with a different colored fluorsecent tag. This technology allows _____s of nucleotides to be determined in a few hours

Answer 27

1000s