Chapter 12: Genomes and Biotechnology

Question 1

What machinery is involved in DNA Replication?

Answer 1

RNA Primers: short strand that codes for synthesis start

Helicase: unwinds DNA complex

Topoisomerase II: relieves stress of unwinding

DNA polymerase: extends an RNA primer by adding to 3’

Question 2

What occurs during DNA Polymerase proofreading?

Answer 2

DNA Polymerase reads the newly added base before adding the next one

Question 3

What is the structure and function of the “Telomerase Enzyme”? Why is it important?

Answer 3

Contains an RNA template that allows the template strand to be lengthened by telomerase repeats. PREVENTS SHORTENING OF DNA after successive replications.

Question 4

What are “Okazaki Fragments” and where do they appear?

Answer 4

Short fragments of DNA that appear on the lagging strand
(synthesized 3’->5’ off of one of the template strands)

Question 5

What are some of the differences between Circular and Linear DNA replication?

Answer 5

Circular (Prokaryotic) DNA Replication:
- one origin of replication
- moves around circle in both directions

Linear (Eukaryotic) DNA Replication:
- multiple origins of replication
- replication bubbles expand until they create one large bubble

Question 6

What is “Polymerase Chain Reaction” (PCR)?

Answer 6

A common method for making copies of a piece of DNA
(selective and highly sensitive)

REPLICATES A LOT OF DNA! (heating up DNA makes the two strands separate. After DNA cools down, the PCR machine introduces 4 essential components which are used to replicate DNA)

Question 7

What are the 4 essential components of PCR (polymerase chain reaction)?

Answer 7

1) Template DNA
(At least one molecule of double-stranded DNA containing the region to be amplified)

2) DNA polymerase
(The enzyme used to replicate the DNA)

3) All four nucleotides
(with the bases A, T, G, or C)

4) Two primers
(two short sequences of single-stranded DNA are required for the DNA polymerase to start synthesis)

Question 8

What are the 3 steps PCR uses to replicate DNA?

Answer 8

1) Denaturation: heating the solution in a plastic tube to a temperature just short of boiling so that the individual DNA strands of the template separate (or “denature”) as a result of the breaking of hydrogen bonds between the complementary bases

2) Annealing: begins as the solution is cooled. Because of the great excess of primer molecules, the two primers bind (or “anneal”) to their complementary sequence on the DNA

3) Extension: the solution is heated to the optimal temperature for DNA polymerase and the polymerase elongates (or “extends”) each primer with the deoxynucleoside triphosphates

Question 9

What is the benefit of using DNA polymerase enzymes that are heat-stable during PCR?

Answer 9

Saves a lot of time!

Normal polymerase lose structure and function in high temperatures, but the DNA polymerase enzymes found in Yellowstone’s “thermus aquaticus” can replicate near boiling point, so we use those ones :)

Question 10

What is the purpose of “gel electrophoresis” after performing PCR?

Answer 10

To determine whether or not PCR has yielded the expected product, a researcher must determine the size of the amplified DNA molecules.

Gel electrophoresis separates DNA molecules by size.
- DNA fragments are negatively charged
-> smaller molecules travel faster and go further than the large molecules.

• LARGE ON TOP, SMALL ON BOTTOM

Question 11

What are “Restriction Enzymes” or “Restriction Endonucleases”? Why are they so important?

Answer 11

(normally restrict phage infection among bacteria)

Enzymes that cut DNA in really clever ways at specific “Restriction Sites”

Makes gene editing research possible! By “copying and pasting” human genes into bacteria, we get to see which proteins are made from which genes.

HUGE FOR MOLECULAR BIOLOGY TODAY!

Question 12

What is “Renaturation”? Why is it important and when would it be used?

Answer 12

The base pairing of complementary single-stranded nucleic acids.
- Also known as hybridization
- Opposite of “Denaturation”

• MAKES IT POSSIBLE TO USE SMALL DNA FRAGMENTS AS A “PROBE”
• USED WHEN: we want to know whether a gene in one species is present in the DNA of a related species, but the nucleotide sequence of the gene is unknown.

Question 13

What is a “Probe”? What is the purpose of probes?

Answer 13

A single-stranded sequence of DNA or RNA that is used to identify specific sequences of DNA or RNA. They are designed as complementary to the part of the genome of interest, so that when the probe and the genome are brought together, the probe will hybridize with the target sequence.

Used to determine whether or not a sample of double-stranded DNA molecules contains sequences that are complementary to it.

Question 14

What is “Recombinant DNA Technology?” Why is it useful today?

Answer 14

The capability to isolate genes from one species and introduce them into another.

Recombining DNA molecules from two (or more) different sources into a single molecule.

• It can be used to generate a large quantity of a protein for study or therapeutic use.

Question 15

What is “Reverse Transcriptase” and how is it used in the Recombinant DNA process?

Answer 15

An enzyme that produces a double-stranded DNA molecule from a single-stranded RNA template.

• Used to obtain a “Donor DNA” fragment that contains only the protein-coding region of the desired gene (“Complementary DNA”)

Question 16

What are some other names for genetically engineered organisms?

Answer 16

“Transgenic Organisms”
“Genetically Modified Organisms” (GMOs)

Question 17

What is “DNA Editing”?

Answer 17

Altering the nucleotide sequence of almost any gene in a deliberate, targeted fashion.

“Rewriting” the nucleotide sequence so that specific mutations can be introduced into genes to better understand their function, or mutant versions of genes can be corrected to restore normal function.

• CRISPR mechanism can do this with almost any gene in any kind of cell

Question 18

What is “DNA Sequencing”?

Answer 18

The ability to determine the nucleotide sequence of DNA molecules

Question 19

What is “Sanger Sequencing”? What are the 5 necessary components of this strategy?

Answer 19

A procedure in which chemical termination of daughter strands helps in determining the DNA sequence

1) A small amount of each of the chain terminators (A, G, C, and T)
2) Larger quantities of all four normal nucleotides
3) A DNA primer
4) A DNA template
5) DNA polymerase

Question 20

What are “Dideoxynucleotides” (a.k.a. chain terminators) and how do they help us sequence DNA?

Answer 20

A, T, C, G nucleotides in which the hydroxyl group on the sugar ring is absent (NO 3’ OH)
-> ALLOW FOR VARIOUS LENGTHS OF DAUGHTER STRANDS

• A.K.A. “Chain Terminator”

Question 21

How do we use Sanger Sequencing to determine the desired DNA sequence?

Answer 21

Each of the four dideoxynucleotides is chemically labeled with a different fluorescent dye, and so all four terminators can be present in a single reaction and still be distinguished.

After DNA synthesis is complete, the daughter strands are separated by size with gel electrophoresis.

The smallest daughter molecules migrate fastest and reach the bottom of the gel.

Question 22

What is a “Genome”?

Answer 22

The term “genome” refers to the genetic material of an organism, cell, nucleus, organelle, or virus. Technically, the human genome refers to the DNA in the chromosomes present in a sperm or egg. However, the term is often used informally to mean all of the genetic material in an organism.

Question 23

How are complete genomes sequenced? What is one common method to do so?

Answer 23

By assembling smaller pieces together

• “Shotgun Sequencing”: The sequenced fragments do not originate from a particular gene or region, but from sites scattered randomly across the whole DNA molecule. “Sequence Assembly” is then used.

Question 24

What complicates sequence assembly?

Answer 24

“Repetitive DNA”: Sequences that are repeated

Question 25

What is one method that could be used to reduce cost and increase speed of genome sequencing?

Answer 25

“Nanopore Sequencing”:
A single strand of DNA is passed through a membrane, and the small change in electrical potential as each nucleotide passes through is used to identify each nucleotide.

Question 26

Why would detailed knowledge of someone’s personal genome be valuable?

Answer 26

“PERSONALIZED MEDICINE”:
An approach in which the treatment is matched to the patient, not the disease. Examination of an individual’s genome sequence, by revealing his or her disease susceptibilities and drug sensitivities, allows treatments to be tailored to that individual. (A.K.A. Precision Medicine)

Question 27

What is “Genome Annotation”?

Answer 27

The process of identifying functional elements along the sequence of a genome, thus giving meaning to it.

Question 28

What are “Sequence Motifs” and why do we utilize them?

Answer 28

Indicative sequences of nucleotides that indicate what types of function (or absence of function) may be encoded in a particular region of the genome.

• We use them because genome annotation is essentially pattern recognition. Researchers begin annotating a genome by identifying patterns (by identifying Sequence Motifs)

Question 29

What are 3 common Sequence Motifs?

Answer 29

1) Open Reading Frame (ORF)
2) Noncoding RNA Molecule
3) Transcription Factor Binding Sites

Question 30

What is an “Open Reading Frame” (ORF)?

Answer 30

A stretch of DNA or RNA consisting of codons for amino acids uninterrupted by a stop codon. In genome annotation, this sequence motif identifies the region as POTENTIALLY PROTEIN CODING.

Question 31

What is the “Transcriptome”?

Answer 31

The complete set of mRNA sequences (or in some cases RNA transcripts) produced by a defined cell type, tissue, or organism.

Question 32

Why is Genome Annotation so important?

Answer 32

An annotated genome summarizes knowledge, guides research, and reveals evolutionary relationships among organisms.

Question 33

What is a “Southern Blot”?

Answer 33

Gel Electrophoresis for DNA Fingerprinting

Question 34

What is a “Western Blot”?

Answer 34

Gel Electrophoresis for Proteins

Question 35

What is a “Northern Blot”?

Answer 35

Gel Electrophoresis for RNA