DNA and DNA related-Processes

Question 1

What is the difference between a nucleoside and a nucleotide?

Answer 1

A nucleotide has a phosphate group attached to a nucleoside.

• A nucleoside is a nitrogenous base and a pentose (ribose) bound together at the pentose’s C-1’

Question 2

What are Proto-oncogenes?

Answer 2

Proto-oncogenes are genes before they undergo their mutation into an oncogene.

Question 3

What types of DNA damage are their?

Answer 3

DNA damage can be:

1. Due to exposure to chemicals or radiation
2. Damage to the DNA backbone
3. Alteration of bases
4. Incorrect base pairing during replication

Question 4

What are oncogenes?

Answer 4

Oncogenes are mutated genes within DNA that cause cancer. Oncogenes primarily encode cell cycle-related proteins.

Question 5

What are the primary functions of DNA polymerase?

Answer 5

The primary function of DNA polymerase is to add DNA nucleotides during replication, asset with mitochondrial DNA replication, and repair DNA.

Question 6

What are centromeres?

Answer 6

Centromeres are regions of heterochromatin that separate the arms of chromosomes. They also join the sister chromatids (one half the side of chromosomes) from the S1 Phase until anaphase in cell devision.

Question 7

What are anti-oncogenes?

Answer 7

Anti-oncogenes are tumor suppressor genes.

Question 8

How many histone proteins are in eukaryotic cells and what is the structure?

Answer 8

There are two copies of histone proteins H2A, H2B, H3, and H4 which form the octamer histone core. DNA wraps around the histone core to form a nucleosome and H1 seals off the DNA as it enters and exits the nucleosome.

Question 9

The efficiency of amplication in PCR in later cycles is reduced due to which of the following causes?

1. Reduction in substrate concentrations
2. Insufficient enzyme and time to synthesize mass quantitites of DNA
3. Build up of PCR product which competes with primeers for hybrid formation
4. All of the above

Answer 9

All of the above

Question 10

What are the steps to clone a gene?

Answer 10

1. Choose a Plasmid
2. Treat the plasmid and the gene to be inserted with a restriction enzyme
   
   • Restriction enzymes are produced by bacteria that recognize and cut nucleic acids at very specific sequences
3. The “sticky ends” of the plasmid and gene stick together

Question 11

What is hybridization?

Answer 11

Hybridization is annealing; the joining of two nucleic acid strands into a single complex.

Question 12

What would be the most likely result of a mutation that inhibits telomerase?

Answer 12

Daughter strands of DNA will have progressively shorter sequences.

Question 13

What are the steps for 1 cycle of a Polymerase Chain Reaction (PCR)?

Answer 13

1. Denaturation
   
   • Ideal conditions occur at 94-96°C
2. Hybridization (annealing)
   
   • Ideal conditions occur at 50-65°C
   • Primer binds to parent strand (cannot do so at high temps because no hydrogen bonding between primer’s nucleotides and DNA strand can occur)
   • (taq) Polymerase then binds to annealed strand to initiate 3 step.
3. Replication (Elongation)
   
   • Ideal conditions occur at 70-80

These steps are repeated in order to achieve the desired amount of DNA strands amplified. 1 cycle = 3 steps occuring once.

Question 14

What is chromatin?

Answer 14

DNA wrapped around histones. All of the DNA coiled around histones collectively forms chromosomes.

Question 15

What causes thymine dimers and what issues do thymine dimers cause?

Answer 15

Ultraviolet light/radiation induces the formation of thymine dimers between adjacent thymine residues in DNA. Thymine dimers causes a bulge on one side of the DNA interfering with DNA replication, normal gene expression, and the DNA double helix structure.

• Thymine dimers are repaired via nucleotide excision repair (NER).

Question 16

Explain the difference between euchromatin and heterochromatin.

Answer 16

Euchromatin is uncondensed (unwound from the histone) during interphase allowing the DNA to be replicated/transcribed. It appears light under light microscopy. Heterochromatin is condensed during interphase (DNA wound around the histone) and is transcriptionally silent/inactive. It appears dark under light microscopy.

Question 17

What is nucleotide excision repair and what does it do?

Answer 17

Nucleotide excision repair (NER) eliminates lesions that are large enough to distort DNA’s double helix structure. There are 5 steps:

1. Recognize the lesion (bulge)
2. Excision endonuclease nicks the DNA surrounding the lesion (on both sides
3. Defective oligonucleotide is removed
4. DNA polymerase fills in the gap with the correct base pairs.
5. DNA ligase seals the nick

Question 18

What is the super structure of DNA?

Answer 18

DNA wraps around the histone core forming a nucleosome. The nucleosomes then stack together forming chromatin.

Question 19

What must occur before DNA polymerase can add base pairs?

Answer 19

RNA primers must be removed before DNA polymerase can perform its job.

Question 20

What assay can be performed to determine the length of a segment of DNA?

Answer 20

Gel electrophoresis allows for determination of the length of a segment of DNA.

• The largest piece of DNA moves the slowest.
• All the DNA moves through the agarose gel due to the negatively charged phospodiester backbone of DNA.
• A current runs through the agarose gel. DNA starts on the negative end which repels DNA and the positive end attracts the DNA.

Question 21

What function do single-strand binding proteins (SSBPs) serve?

Answer 21

They prevent the separated strands of DNA from re-annealing during replication and transcription.

Question 22

What are telomeres?

Answer 22

Telomeres prevent essential DNA from being lost during replication. The are simple repeating sequences (TTAGGG) that are replaced/repaired by telomerase.

• Since telomeres contain high C-G content, they serve a second function by preventing the DNA from unraveling at the end.

Question 23

What is DNA Primase’s function?

Answer 23

DNA Primase adds RNA primers which allow DNA polymerase to bind and begin replicating a complimentary daughter strand.

Question 24

What is a PCR (Polymerase Chain Reaction)?

Answer 24

Polymerase Chain Reaction (PCR) is a process that amplifies a small piece of DNA exponentially.

After (n) cycles there will be 2ⁿ daughter strands:

Ex: 2³⁰ = 1,073,741,824 daughter strands

Question 25

What are the different types of DNA Polymerase in Eukaryotes and Prokaryotes, and what are their primary function?

Answer 25

Question 26

What direction does DNA polymerase read and add base pairs?

Answer 26

DNA polymerase reads and adds base pairs in the 5' to 3' direction.

Question 27

What is a nucleosome?

Answer 27

A nucleosome is the DNA (approximately 150 base pairs) wrapped around the histone protein complex.

Question 28

What does metastasis mean and how is it related to cancer?

Answer 28

Metastasis means migration. Cancer cells can break off from the other cancer cells and migrate to another place in the body. Once the cancer cell settles down (stops migrating), it can begin to replicate and infect that area of the body.

Question 29

What is recombinant DNA?

Answer 29

Recombinant DNA is a process through which human gene products (hormones, proteins, etc.) are produced by amplifying a specific portion of a human gene and inserting that code into microbial plasmids for production. * Recombination in short, refers to inserting foreign DNA into a host plasmid.

Question 30

Why is the lagging strand more likely to have mutations?

Answer 30

The lagging strand is more likely to have mutations because it must constantly start and stop the process of DNA replication. Additionally, it contains many more RNA primers, all of which must be removed and filled in with DNA. * DNA ligase lacks proofreading ability contributing to why the lagging strand has more mutations as part of the DNA polymerase enzyme proofreads.

Question 31

What does southern blotting test?

Answer 31

Southern blotting tests for the presence of a specific segment of DNA or RNA

Question 32

Upon cloning recombinant DNA into a host plasmid, you want to confirm that the recombinant has been inserted into the plasmid correctly. Which technique would you use to confirm this?

Answer 32

Gel electrophoresis will separate DNA by size. You will be able to clearly see if your recombinant has been inserted into the plasmid because it will migrate slower than the plasmid.

Question 33

How does DNA polymerase recognize which is the template strand and once the daughter strand is synthesized?

Answer 33

The parent strand is more heavily methylated, whereas the daughter strand is barely methylated at all. This allows DNA polymerase to distinguish between the two strands during proofreading.

Question 34

What is the key structural difference between the types of lesions corrected by nucleotide excision repair vs those corrected by base excision repair?

Answer 34

Nucleotide excision repair corrects lesions that are large enough to distort the DNA double helix. Base Excision repair corrects lesions that are small enough to not distort the DNA double helix.

Question 35

What is one of the main issues that can occur with randomly integrated DNA via gene therapy?

Answer 35

Randomly integrated DNA poses a risk of integrating near and activating a host oncogene (inducing cancer). * Ex: Among the children treated with gene therapy for SCID, a small number have developed leukemias (cancers of white blood cells).

Question 36

What type of DNA library would be beneficial for which of the following studies? 1. DNA studies for introns, centromeres, and telomeres? 2. DNA studies used to express recombinant proteins or to perform gene therapy?

Answer 36

Genomic libraries (non-coding regions included) are beneficial for DNA studies observing introns, centromeres, or telomeres. cDNA libraries (expressed genes only) are beneficial for DNA studies used to express recombinant proteins and gene therapy.

Question 37

What does PCR accomplish for a researcher?

Answer 37

PCR creates significant amount of copies of a particular DNA sequence from a sample containing very few copies of DNA.

Question 38

What does Southern Blotting accomplish for a researcher?

Answer 38

Southern blotting is useful when searching for a particular DNA sequence because it separates DNA fragments by length and then probes for a sequence of interest. * Radioactive molecules/atoms are utilized to produce fluorescence.

Question 39

What is the difference between knockout and transgenic mice?

Answer 39

Transgenic mice have a gene introduced into their germ line or embyronic stem cells to observe the effects of that gene. Therefore, transgenic mice are best suited for studying the effects of dominant alleles. Knockout mice are those in which a gene of interest has been removed, rather than added.