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. page 248
DNA replication is semiconservative, meaning that each daughter DNA molecule consists of a newly synthesized strand and a strand that was present in the parental DNA molecule. page 248
Nucleotides are added to the 3′ end of the growing strand. Therefore, synthesis proceeds in a 5′-to-
At the replication fork, one new strand is synthesized continuously (the leading strand) and the other is synthesized in small pieces that are ligated together (the lagging strand). page 251
DNA polymerase requires RNA primers for DNA synthesis. page 252
DNA polymerase can correct its own mistakes by detecting a pairing mismatch between a template base and an incorrect new base. page 254
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. page 254
Telomerase prevents chromosomes from shortening after each round of replication by adding a short stretch of DNA to the ends of chromosomes. page 255
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. page 257
PCR requires a DNA template, DNA polymerase, the four nucleoside triphosphates, and two primers. It is a repeated cycle of denaturation, annealing, and extension. page 259
Gel electrophoresis allows DNA fragments to be separated according to size, with small fragments migrating farther than big fragments in a gel. page 259
Restriction enzymes cut DNA at specific recognition sequences called restriction sites, which cut DNA leaving a single-
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. page 261
A Southern blot involves using a filter paper as a substrate for DNA fragments that have been cut up by restriction enzymes and hybridized to a probe. page 262
In Sanger sequencing of DNA, dideoxynucleotide 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. page 263
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. page 264
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12.4 Genetic engineering allows researchers to alter DNA Sequences.
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. page 264
Recombinant DNA is the basis for genetically modified organisms (GMOs), which offer both potential benefits and risks. page 266
Almost any DNA sequence in an organism can be altered by means of a form of DNA editing called CRISPR, which uses modified forms of molecules found in bacteria and archaeons that can cleave double-
Explain how DNA structure itself suggests a mechanism of DNA replication.
DNA consists of two antiparallel strands, meaning that the 3' hydroxyl end of one strand is opposite the 5' phosphate group of the other strand. In the antiparallel helical coil, a purine base (A or G) of one strand base-
Explain how the chemical structure of deoxynucleotides determines the orientation of the DNA strands and how this affects the direction of DNA synthesis.
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 in the 5' to 3' direction.
List the differences and similarities in the way the two daughter strands of DNA are synthesized at a replication fork.
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.
Explain why replicating the tips of linear chromosomes is problematic and how the cell overcomes this challenge.
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 a 100-
Describe what PCR does. Name and explain its three steps and give at least two uses for the PCR technique.
PCR is used to generate many copies of a piece of DNA. The three steps of PCR are (1) denaturation of the double-
Explain how the properties of DNA determine how it moves through a gel, is cut by restriction enzymes, and hybridizes to other DNA strands.
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.
Describe how DNA molecules are sequenced.
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-
Describe how recombinant DNA techniques can be used to express a mammalian gene in bacteria.
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 that also replicates the donor, mammalian gene, at the same time.
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