hlw_ch12

CHAPTER SUMMARY

12.1 IN DNA REPLICATION, A SINGLE PARENTAL MOLECULE OF DNA PRODUCES TWO DAUGHTER MOLECULES.

DNA replication involves the separation of the two strands of the double helix at a replication fork and the use of these strands as templates to direct the synthesis of new strands.
Nucleotides are added to the 3' end of the growing strand. Therefore, synthesis proceeds in a 5'-to-3' direction.
At the replication fork, one new strand is synthesized continuously (the leading strand) and the other is synthesized in small pieces (the lagging strand).
An RNA primer is required to begin DNA synthesis.
DNA polymerase can correct its own mistakes by detecting a pairing mismatch between a template base and an incorrect new base.

12.2 THE REPLICATION OF LINEAR CHROMOSOMAL DNA REQUIRES MECHANISMS THAT ENSURE EFFICIENT AND COMPLETE REPLICATION.

Chromosomal DNA has many origins of replication and replication proceeds from all of these almost simultaneously.
Telomerase prevents chromosomes from shortening after each round of replication by adding a short stretch of DNA to the ends of chromosomes.

12.3 TECHNIQUES FOR MANIPULATING DNA FOLLOW FROM THE BASICS OF DNA STRUCTURE AND REPLICATION.

The polymerase chain reaction (PCR) is a technique for amplifying a segment of DNA.
PCR requires a DNA template, DNA polymerase, the four nucleoside triphosphates, and two primers. It is a repeated cycle of denaturation, annealing, and extension.
Gel electrophoresis allows DNA to be separated according to size, with small fragments running farther than big fragments in a gel.
Restriction enzymes cut DNA at specific recognition sequences called restriction sites.
Restriction sites are usually palindromic, meaning that the recognition sequence is the same on each strand when read in a given direction. Restriction enzymes cut DNA leaving a single-stranded overhang or a blunt end.
In DNA denaturation, the two strands of a single DNA molecule separate from each other. In DNA renaturation or hybridization, two complementary strands come back together again.
A Southern blot involves cutting up DNA molecules using restriction enzymes, separating the resulting DNA fragments by size using gel electrophoresis, denaturing the DNA fragments in the gel, transferring the DNA fragments to filter paper, and detecting fragments of interest by allowing renaturation with a labeled probe.
In Sanger sequencing of DNA, chain terminators are used to stop the DNA synthesis reaction and produce a series of short DNA fragments from which the DNA sequence can be determined.
New DNA sequencing technologies are being developed to increase the speed and decrease the cost of sequencing, perhaps making it possible to sequence everyone’s personal genomes.

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12.4 RECOMBINANT DNA TECHNOLOGY COMBINES DNA FROM TWO OR MORE ORGANISMS.

A recombinant DNA molecule can be made by cutting DNA from two organisms with the same restriction enzyme and then using DNA ligase to join them.
Recombinant DNA is the basis for genetically modified organisms (GMOs), which offer both potential benefits and risks.

Self-Assessment Question 1

Explain how DNA structure relates to DNA replication.

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Model Answer:

DNA consists of two antiparallel strands, meaning that the 3’ hydroxyl end of one strand interacts with the 5’ phosphate group of the other strand. In the antiparallel helical coil, a purine base (A or G) of one strand interacts with a pyrimidine base (T or C, respectively) of the other strand. This mechanism allows the two strands to have identical genetic information in the form of nucleotide sequence. When DNA replication occurs, the two strands separate (“unzip”) from each other and both are used as a template for the replication of two new DNA strands.

Self-Assessment Question 2

Describe the orientation of the two DNA strands and the direction of DNA synthesis.

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Model Answer:

The orientation of the two DNA strands is antiparallel. This means that the 3’ hydroxyl end of one strand is opposite the 5’ phosphate group of the other strand. When the two strands separate and DNA replication begins, nucleotides are added to the 3’ end of both strands so DNA replication occurs 5’ to 3’.

Self-Assessment Question 3

List the differences and similarities in synthesis of the two daughter strands of DNA.

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Model Answer:

The similarities of the resulting daughter strands from DNA replication are that they will encode the same genetic information in the form of nucleotide sequence and they will also be paired with one of the original parental DNA strands. The two strands are different in the way that they are replicated although the chemistry of strand elongation is the same for both. On both strands, replication occurs in the 5’ to 3’ direction.

The leading strand has the 3’ end of its DNA pointed toward the replication fork and thus will be synthesized as one long, continuous polymer. The replication of the other strand, or lagging strand, is a little more complex due to its 5’ end pointing toward the replication fork. Since DNA can only be replicated in the direction of 5’ to 3’, the lagging strand is synthesized in short, discontinuous pieces. Each new piece, or Okazaki fragment, is elongated at its 3’ end until it reaches the piece in front of it.

Self-Assessment Question 4

Explain why replicating the tips of linear chromosomes is problematic and how the cell overcomes this challenge.

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Model Answer:

Replicating the tips of linear chromosomes is problematic because during the synthesis of the lagging strand, about 100 base pairs at the 3’ end are not replicated. This is due to the fact that the sequence of the last RNA primer binding site is not replicated because the primer is bound at the time of replication and then removed when replication is finished. This results in about 100 base pair gap in sequence This loss of sequence is restored through an enzyme called telomerase, which is most active in germ and stem cells. Eukaryotic chromosomes are capped by a repeating sequence called the telomere, which does not encode any genes. When 100 nucleotides of the telomere are lost, the telomerase replaces them. This shortening and lengthening of the chromosome is not detrimental to the cell because there are no coded genes in this region.

Self-Assessment Question 5

Name the three steps of PCR and at least two uses for the PCR technique.

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Model Answer:

The three steps of PCR are (1) denaturation of the double-stranded DNA into two individual strands, (2) annealing of the two primers to their complementary sequence on the DNA template strands, and (3) extension of the parental DNA strands through elongation (5’ to 3’) by DNA polymerase (by extending the primers). PCR can be used in a variety of ways such as DNA fingerprinting, where a person’s DNA is sequenced and potentially matched to evidence found at a crime scene (paternity tests are performed in a similar way). PCR can also be used to identify an organism based on a known conserved region of their DNA, and it can also be used to mass-produce certain sequences of DNA for DNA-based vaccines.

Self-Assessment Question 6

Explain how the properties of DNA determine how it moves through a gel, is cut by restriction enzymes, and hybridizes to other DNA strands.

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Model Answer:

Since fragments of DNA are negatively charged, pieces will move through a porous gel when an electric current is passed through it. The distance the DNA pieces move through the gel is based on their size, with larger fragments moving more slowly and smaller fragments moving through the gel quickly. To get these fragments of DNA, restriction enzymes, which each recognize a particular sequence of DNA (the restriction site), cleave the DNA at its specified site. DNA can also hybridize to other DNA strands if their nucleotide sequences are complementary to each other.

Self-Assessment Question 7

Describe how DNA molecules are sequenced.

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Model Answer:

In Sanger sequencing, the sequence of a template DNA strand is unknown. This DNA is used as the template for replication by DNA polymerase. A DNA primer, polymerase, normal nucleotides and chain terminating nucleotides are all added to the template and replication of the unknown strand begins. Elongation of this strand stops whenever a dideoxynucleotide terminator (a nucleotide in which the 3’ hydroxyl group on the sugar ring is absent) is incorporated at the 3’ end. The four altered nucleotides are chain terminators, each labeled with a different fluorescent dye, so the result of Sanger sequencing is a tube filled with different length fragments of the same DNA sequence. The mixture is then run on a gel and “read” by looking at the fluorescent pattern of the dideoxynucleotides incorporated into the sequence. For example, a sequence of 5’-ATGC-3’ would be “read” as green-red-blue- purple (for the fluorescent dyes).

Self-Assessment Question 8

Describe how recombinant DNA techniques can be used to express a mammalian gene in bacteria.

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Model Answer:

The mammalian gene (donor DNA) can be expressed in a bacterium through its insertion into a vector DNA that can be replicated in the bacterium. The same restriction enzymes are used to cut the donor DNA and the vector DNA so that they now have complementary ends. A ligation reaction is then performed to insert the donor DNA into the vector DNA. The vector DNA is then inserted into the bacterium and replicated through the normal method which also replicates the donor, mammalian gene, at the same time.