Chapter 8 and 9

Question 1

What is Chargaff’s ratio?

Answer 1

The complimentary base pairs have the same ratio as each other

(ex: if 20% A, then there is also 20% T)

Question 2

Adenine pairs with _____ and Guanine pairs with ______

Answer 2

Thymine
Cytosine

Question 3

In RNA, Uracil replaces _____ and pairs with ______

Answer 3

Thymine
Adenine

Question 4

What are the four forces that stabilize the DNA double helix?

Answer 4

Hydrophobic effect (all the non-polar pieces of the helix are on the inside)
Stacking interactions between the base pairs
H-bonding between the base pairs
Charge-charge interactions

Question 5

What is magnesium’s role in stabilizing the double helix?

Answer 5

Mg2+ bonds with the negative phosphates on the helix backbone and decreases the amount of electron repulsion from the phosphates

Question 6

In DNA, the ribose sugar that serves as the nucleotide base is always in the ______ form

Answer 6

Beta-furanose

Question 7

_______ and ______ are purine bases while ______, _______, and ______ are pyrimidine bases

Answer 7

Adenine and Guanine

Thymine, Uracil and Cytosine

Question 8

All nucleotide bases absorb around ____ nm on the UV spectrum

Answer 8

260

Question 9

Why do the bases stack well on the inside of the DNA helix?

Answer 9

When they are bonded to their complimentary base, G&C have the same shape as A&T (isosteric). This makes them good for stacking because they can fit on top of each other

Question 10

The helical pitch of DNA’s B form repeats every ____ base pairs

Answer 10

10

Question 11

Why does double stranded DNA have less absorbance than single strand DNA on a UV spectrum?

Answer 11

In double stranded DNA, the base pairs are less accessible because they are inside of the phosphate backbone. This is not an issue for single strand DNA because the base pairs are not bonded to anything, so they are always accessible

Question 12

When looking at a graph of melting temperatures (Tm), what bonds would you expect to be present in a sequence that has a high Tm?

Answer 12

Since h-bonds are hard to break, something with a higher Tm must have a lot of h-bonds. This means there must be a lot of C&G present, since they pair with 3 h-bonds as opposed to A&T that pair with 2 h-bonds

Question 13

dNTP stands for

Answer 13

deoxyribonucleotide tri-phosphate

Question 14

What is the difference between a nucleoside and a nucleotide?

Answer 14

Nucleosides are made up of the pentose (ribose) and a nitrogenous base (A,G,C,T)

Nucleotides are the same as nucleosides, but they also have a phosphate group attached to C5

Question 15

What is the purpose of mutating DNA and RNA? Ex. methylating, epigenetics

Answer 15

Mutating/modifying can happen at either the DNA or protein level and can change the way genes are expressed. This can be beneficial for things like RNA vaccines by helping the body to recgonize them differently, a way to distinguish native DNA from foreign DNA, or to turn genes on and off

Question 16

What occurs during base deamination and what are the resulting effects?

Answer 16

Deamination occurs when bases lose their exocyclic amino groups. This happens naturally and the body can compensate, but when it begins to occur at too fast of a rate it can be mutagenic. Ex: if cytosine turns into uracil, the body will recognize it as an RNA strand and will cut that section out of the DNA

Overall, deamination makes mutations that can affect the expression of the gene by causing things to pair up differently than they should be

Question 17

What does it mean that DNA is antiparallel?

Answer 17

The strands are not identical but complimentary, so they pair up in 5’-3’ and 3’-5’

Question 18

What is depurination?

Answer 18

Occurs when the bond between the base and the pentose hydrolyzes and the two separate. This happens much faster for purines than pyrimidines, which is why it’s called depurination

Question 19

What is the danger of base crosslinking?

Answer 19

Crosslinking- when a base bond with the base above or below it instead of the base across from it

Crosslinking can put kinks in the DNA. This happens naturally when we are exposed to the sun and the body can handle it on that scale, but when the cross linking begins to occur too fast it can cause fragmentation or ring opening, and ultimately permanent mutation that can cause things like cancer

Question 20

What can happen when base pairs are methylated?

Answer 20

Methylation can be mutagenic, carcinogenic or cytotoxic. This is because it can change the bonding patterns of the base pairs and create bonds that should not appear (like G with T)

Question 21

What is the process of natural DNA denaturing/annealing?

Answer 21

When temperatures get too high, DNA will denature. If the temperature goes back down, the DNA will anneal- meaning it will come back together in the double helix. This occurs when DNA is replicating, since the parent strands have to denature to serve as templates while they make the daughter strands

Question 22

What are some other functions of nucleotides (besides serving in DNA)?

Answer 22

Can serve as energy as ATP, make up structural components of enzyme cofactors, and act as allosteric effectors

Question 23

What is the process of the Sanger Method for sequencing DNA? (Dideoxy Method)

Answer 23

First, the template DNA is added to four test tubes along with a primer, polymerase, the four dNTP’s and one of the ddNTP’s per test tube.

The dNTP’s will allow for polymerization to continue because they are the regular nucleotides, but the ddNTP’s lack the OH needed for polymerization, so the strand will stop replicating when the ddNTP attaches.

The strands are then placed in electrophoresis and the complimentary strand is read from the gel (the bases are placed in alphabetical order on the gel –> A,C,G,T)

Question 24

What is the automated DNA sequencing method?

Answer 24

A primer is added to the original DNA strand along with dNTP’s and a ddNTP. Like the Sanger method, the DNA will replicate until the ddNTP attaches, then the strands will be put into capillary gel and their fluorescent tags are read with a laser as they go through electrophoresis. This creates a graph that will then give peaks corresponding to each nucleotide.

For larger sequences, the DNA is broken into sections much like AA sequencing

Question 25

What is the process of DNA cloning?

Answer 25

First, a specific gene needs to be amplified with a primer, dNTP, polymerase and Mg2+ for stabilization.

The amplified gene is then placed inside a plasmid that has been cut with restriction endonucleases. Ligases glue the cut ends together so that the plasmid can close properly, then the entire thing is put inside a bacteria so it can be replicated over and over

Question 26

Restriction endonucleases are normally found in bacteria, where they _____

Answer 26

Cut foreign DNA coming into the bacteria, like from a bacteriophage

Question 27

Restriction endonucleases can make staggered cuts known as ____ ends, or straight cuts known as ____ ends

Answer 27

Sticky

Blunt

Question 28

What five things are needed in PCR?

Answer 28

Template DNA
Primer
dNTP
Mg2+
Polymerase (that can withstand high temperatures)

Question 29

How does PCR work?

Answer 29

DNA is first denatured at high temperatures, then primer is attached and begins to replicate the strands. Once the replication is done, the DNA is denatured again and the primer is added to the new strands. This continues for about 40 copies until the DNA has been amplified enough to be read for the specific gene of interest

Question 30

What is recombination and what is its purpose?

Answer 30

Recombination occurs when a gene is replaced by a mutated copy. This is done in things like GMO foods where foreign DNA is inserted into the organism and needs to be recombined with the original DNA to prevent the organism from destroying the foreign DNA

Question 31

What is site directed mutagenesis?

Answer 31

An AA can be mutated by changing the DNA present so that the mutated gene is expressed instead. This is done with special primers that are incorporated into DNA