MCB chapter 12 DNA replication and mainpulation

leading strand

- the daughter strand -A daughter strand that has its 3′ end pointed toward the replication fork, so as the parental double helix unwinds, this daughter strand can be synthesized as one long, continuous polymer.

The process of the synthesis and removal of RNA primers in the lagging strand

1. RNA primase lays down and RNA primer 2. DNA polmerase extends the RNA primer 3. A different DNA polymerase removes the primer and replaces it with DNA. 4. DNA ligase forms a bond joining the two DNA fragments.

a new DNA strand can only be elongated at which end

3' end

You run a PCR reaction for five cycles starting with a single DNA duplex. Theoretically, how many copies of your sequence would you now have? 8 12 16 24 32

32

gel electrophoresis

A procedure to determine the size of a DNA fragment, in which DNA samples are inserted into slots or wells in a gel and a current passed through. Fragments move toward the positive pole according to size.

DNA polymerase

An enzyme that is a critical component of a large protein complex that carries out DNA replication. -all DNA polymerases share the same basic function in that they synthesize a new DNA strand from an existing template. -Most, but not all, also correct mistakes in replication

What is the benefit of using Taq polymerase in PCR? Because it is taken from bacteria that live in high temperatures, it doesn't have a proofreading function. Because it is taken from bacteria, this enzyme works much more efficiently than other types of DNA polymerase. Because it is taken from bacteria that live in high temperatures, it stays active during denaturation steps of the reaction Because it is taken from bacteria, it makes fewer mistakes. None of the answer options is correct.

Because it is taken from bacteria that live in high temperatures, it stays active during denaturation steps of the reaction

Which of the following is true regarding DNA replication? Both strands get shortened at opposite ends during replication. Both strands get shortened at the same end during replication. Only the leading strand gets shortened during replication. Only the lagging strand gets shortened after every other round of replication. Only the lagging strand gets shortened during replication.

Both strands get shortened at opposite ends during replication.

Which of the following enzymes is necessary to produce recombinant DNA? helicase Taq polymerase topoisomerase primase DNA ligase

DNA ligase

requires both a template and a free 3′ end to elongate a nucleotide chain. RNA primase Helicase Topoisomerase II DNA polymerase

DNA polymerase

Why are primers needed for DNA replication? DNA polymerase can only add nucleotides to an existing chain. A tiny amount of RNA is needed to tell the cell where genes are located. The primers help with the proofreading function of DNA polymerase. They help with the joining of Okazaki fragments. They help direct the placement of the telomeres.

DNA polymerase can only add nucleotides to an existing chain.

moves outward from the origin of replication and breaks hydrogen bonds between DNA nucleotides. RNA primase Helicase Topoisomerase II DNA polymerase

Helicase

Cutting DNA molecules allows pieces from the same or different organisms to be brought together in recombinant DNA technology,

It also is a way to determine whether or not specific sequences are present in a segment of DNA, as techniques for cutting DNA depend on specific DNA sequences. Finally, cutting DNA allows whole genomes to be broken up into smaller pieces for further analysis, such as DNA sequencing. The method for cutting DNA makes use of a class of enzyme that recognizes specific, short nucleotide sequences in double-stranded DNA and cleaves the DNA at these sites. The enzymes are known as restriction enzymes, of which about 1000 different kinds have been isolated from bacteria and other microorganisms. The recognition sequences the enzymes cleave, often called restriction sites, are typically four or six base pairs long. Most restriction enzymes cleave double-stranded DNA at or near the restriction site. For example, the enzyme EcoRI has the following restriction site:

is the first enzyme to synthesize a nucleic acid at a replication fork. Helicase RNA primase Topoisomerase II DNA polymerase

RNA primase

Which of the following restriction enzymes produce sticky ends? Select all that apply. (An arrow indicates the restriction site.) Eco53kl with the restriction sequence 5′- GAG↓CTC-3′ AbsI with the restriction sequence 5′- CC↓TCGAGG-3′ BssKI with the restriction sequence 5′- ↓CCCGGG-3′ TaiI with the restriction sequence 5′- ACGT↓ -3′ SspI with the restriction sequence 5′- AAT↓ATT-3′

TaiI with the restriction sequence 5′- ACGT↓ -3′

Which of the following statements is true regarding telomeres? Telomeres contain non-essential genes that are not necessary for cell survival. Telomeres are typically only a few base pairs in size. Telomeres are enzymes that repair telomerases (which cap the end of linear chromosomes). Telomeres can contain repeated base sequences. Telomeres are repaired to the same extent in germ cells and somatic cells.

Telomeres can contain repeated base sequences.

renaturation

The base pairing of complementary single-stranded nucleic acids to form a duplex; also known as hybridization, it is the opposite of denaturation.

What would happen if telomerase stopped working in a cell in which it normally functions at high levels? The cells would be able to divide indefinitely. Cancer would be the result. The cell would die immediately. The cell would eventually die as the DNA continued to shorten. None of the answer options is correct.

The cell would eventually die as the DNA continued to shorten.

You do a PCR reaction, run a sample of the product on a gel, and use a dye to visualize the bands. You expect a single product of 300 bp, which you observe, but you also see a band of about 550 bp. Can you suggest a reason why? Does the size of the unexpected band indicate anything about your target sequence?

The most likely reason for observing an unexpected PCR product is that the primers annealed not only to sites flanking the target region but also annealed in the correct orientation to sites flanking a different, nontarget region of the genome. The size of the nontarget region is completely unpredictable, so there is nothing significant in the size of the unexpected product being 550 bp.

You have determined that the newly synthesized strand of DNA in your sequencing reaction has the sequence 5′-ACTACCGAGT-3′. What is the sequence of the template strand?

The sequence of the template strand is antiparallel to the synthesized strand and inferred from complementary base pairing as 5′-ACTCGGTAGT-3′.

In recombinant DNA experiments, ______ is used to cut pieces of DNA, and ______ joins these segments to form recombinant DNA. a restriction enzyme; DNA ligase a RNA polymerase; a restriction enzyme a DNA polymerase; DNA ligase DNA ligase; a restriction enzyme DNA ligase; a plasmid

a restriction enzyme; DNA ligase

Which of the following reasons explains why bacteria can continually divide? because their DNA is circular so the DNA never shortens after replication because they have plasmids because they are so simple because DNA replication is much quicker in bacteria than in eukaryotes because they only have one chromosome

because their DNA is circular so the DNA never shortens after replication

What is the first thing to occur in DNA replication? Question 1 choices Choice A., the strands of the DNA double helix are separated Choice B., the synthesis of a short RNA primer Choice C., the extension of DNA from the 3′ end of the RNA primer Choice D., the removal of the RNA primer, which is replaced by DNA Submit

choice A

On which strand is DNA synthesis discontinuous, occurring in fragments that are later connected? Question 4 choices Choice A., the leading strand Choice B., the lagging strand Choice C., Both strands synthesize DNA in fragments.

choice B

Which of the strands use a template for DNA replication? Question 2 choices Choice A., the leading strand Choice B., the lagging strand Choice C., Both strands use a template during DNA replication.

choice C. each of the separated parental strands serves as a template for the synthesis of a daughter strand. Production of each daughter strans is initiated by synthesis of a short RNA primer.

Which of the following statements about DNA replication is true? Question 5 choices Choice A., On the leading strand, new DNA is added to the 3′ end of the growing strand, whereas on the lagging strand, new DNA is added to the 5′ end. Choice B., The lagging strand requires an RNA primer to initiate DNA synthesis, whereas the leading strand does not require an RNA primer. Choice C., When DNA polymerase reaches a primer from prior DNA synthesis, the primer is removed and replaced with DNA. Choice D., DNA polymerase performs DNA synthesis on the leading strand, and RNA polymerase performs synthesis on the lagging strand.

choice C. As seen in the lagging strand

What is the name for the enzyme activity that DNA polymerase has when it removes an improperly paired base from the end of the growing strand? Question 3 choices Choice A., helicase activity Choice B., polymerase activity Choice C., exonuclease activity Choice D., endonuclease activity

choice C. it cleaves the improrperly paird base from the growing strand

These actions are all involved in PCR: extension initiation denaturation termination annealing Select the answer that places them in the correct order. initiation, extension, termination denaturation, annealing, extension denaturation, extension, termination initiation, denaturation, extension annealing, initiation, termination

denaturation, annealing, extension

DNA polymerase is the enzyme that separates the two strands of DNA during DNA replication. true false

false

If a restriction site of AatII is 5′-GACGTC -3′ then 3′-GACGTC-5′ is also an AatII restriction site. true false

false

Okazaki fragments are ligated into a continuous DNA molecule after replication is completed. true false

false

Recombinant DNA only occurs when you take DNA from another organism and insert it into a bacterium. true false

false

The semiconservative model of DNA replication means that each DNA strand consists of some newly synthesized DNA and some parental DNA. true false

false

Transformation is the process where DNA is extracted from a plasmid. true false

false

The following enzymes play important roles in DNA replication: DNA polymerase, primase, ligase, helicase. In what order do they work during replication? helicase, primase, DNA polymerase, ligase DNA polymerase, helicase, primase, ligase ligase, helicase, DNA polymerase, primase DNA polymerase, primase, ligase, helicase primase, helicase, ligase, DNA polymerase

helicase, primase, DNA polymerase, ligase

what are the bonds that hold nucleotids together

hydrogen bonds (complementary base pairing)

Okazaki fragments are found on _____ strand of DNA. the parental both the leading and lagging the leading the lagging

lagging

why must the strands of DNA be antipararell

maximize the hydrogen bonding

What is the function of the enzyme DNA polymerase? to synthesize a polypeptide using DNA as a template to synthesize a strand of mRNA using DNA as a template to synthesize a strand of DNA using DNA as a template to synthesize a strand of DNA using a polypeptide as a template to synthesize a strand of mRNA using mRNA as a template

o synthesize a strand of DNA using DNA as a template

^^^^^^ overhanging recombining

see example in notability

In gel electrophoresis, DNA fragments are separated based on their: charge. size. how many adenine bases are in the fragment. sequence. how many thymine bases are in the fragment.

size

DNA replication ^^^^^

the parental strands separate and new complementary partner strands are made— - each indiv parental strand serves as a model (template strand) for the creation of a daughter strand. - the sequence 5′-ATGC-3′ in the template strand specifies the sequence 3′-TACG-5′ in the daughter strand ^^^^^ -The first thing that has to happen is the DNA strand has to unwind. the H bonds are broken. (this is why H bonds are important because they are easy to break) - RNA binds (complementary base pairing)

^^^^ components involved in recombinant DNA of a plasmid

- Donor DNA (use a chromosome) and vectosr (plasmid) DNA are both cleaved with the same restriction enzyme - the donor DNA is then attached to the vector DNA. They are joined by a DNA ligase - it is then put into bacteria - the bacteria then uptake the new plasmid - once into the bacterial cells the bacteria's replication machinery will then see the orgin of replication on the plasmid and then start making replications of that particular plasmid. - The bacteria can also create the protein that is made from that gene sequence of the plamsid. - which allows us to make any protiens we choose. - as the bacterial DNA replicates and divides, the recombinant DNA is also replicated and transferred to the daughter cells. -Basically a factory of cells. - this is used to make insulin for those who are diabetic OVERALL: -DNA encoding the protein is pasted into a plasmid and replicated in the host. The host transcribes and translates the protein. The protein is extracted and purified for use.

proofreading

- a process where most DNA polymerases can correct their own errors by excising and replacing a mismatched base. -DNA polymerase can correct errors because it detects mispairing between the template and the most recently added nucleotide. Mispairing between a base in the parental strand and a newly added base in the daughter strand activates a DNA-cleavage function of DNA polymerase that removes the incorrect nucleotide and inserts the correct one in its place.

How is DNA replicated so quickly

- because in a long DNA molecule, replication begins almost simultaneously at many places. Each point at which DNA synthesis is initiated is called an origin of replication. The opening of the double helix at each origin of replication forms a replication bubble with a replication fork on each side, each with a leading strand and a lagging strand with topoisomerase II, helicase, and single-strand binding protein playing their respective roles. - DNA synthesis takes plact at each replication fork, and as the replication forks move in opposite directions the replication bubble increases in size. When two replication bubbles meet, they fuse to form one larger replication bubble. - When two replication bubbles fuse and the leading strand from one meets the lagging strand from the other, the ends of the strands that meet are joined by DNA ligase, just as happens when the discontinuous fragments within the lagging strand meet DNA molecules that are circular: - typically have only one origin of replication - Replication takes place at both replication forks, and the replication forks proceed in opposite directions around the circle until they meet and fuse on the opposite side, completing one round of replication.

circular DNA molecule VS linear DNA

- can be replicated completely becuase it has no ends and the replication forks can move around the circle. - Linear DNA molecules have ends and each round of replication makes the ends shorter: on the lagging strand, which grows away from the replication fork, many primers are required, and the final RNA primer is synthesized about 100 nucleotides from the 3′ end of the template. Because this primer initiates synthesis of the final Okazaki fragment of the lagging strand, there is no other Okazaki fragment to synthesize the missing 100 base pairs and remove the primer. Therefore, when DNA replication is complete, the new daughter DNA strand (light blue in Fig. 12.11) will be missing about 100 base pairs from the tip (plus a few more owing to the length of the primer). When this daughter strand is itself replicated, the newly synthesized strand must terminate at the shortened end of the template strand, and so the new duplex molecule is shortened by about 100 base pairs from the original parental molecule. The strand shortening in each round of DNA replication is a problem because without some mechanism to restore the tips, the DNA in the chromosome would eventually be nibbled away to nothing. BUT through evoltion, this problem has been solved by the use of TELOMERES. - Each end of a eukaryotic chromosome is capped by a repeating sequence called the telomere. The repeating sequence that constitutes the telomere differs from one group of organisms to the next, but in the chromosomes of humans and other vertebrate it consists of the sequence 3′-GGGATT-5′ repeated over and over again. The telomere is slightly shortened in each round of DNA replication, as shown in Fig. 12.11, but the shortened end is quickly restored by an enzyme known as telomerase, which contains an RNA molecule complementary to the telomere sequence. the telomerase replaces the lost telomere repeats using its RNA molecule as a guide to add successive DNA nucleotides to the 3′ end of the template strand. Once the 3′ end of the template strand has been elongated, an RNA primer and the complementary DNA strand are synthesized. In this way the original telomere is completely restored. Because there are no genes in the telomere, the slight shortening and subsequent restoration that take place have no harmful consequences.

^^^ DNA sequencing

- in DNA sequencing the order of the bases in a DNA stand can be determined by copying short contiguous segments of the strand and then determiing the base at the ned of the segment using fluorescent probes - Sanger sequencing requires a DNA template, one primer, deoxynucleotides, a small amount of dideoxynucleotides, and DNA polymerase. - There is no OH- on a dideoxynucleotide which terminates the DNA sequencing chain. These are able to bet detected on a scanner becuase it uses a fluorophore ( a particular signal that emits light. Certain light is related to a particular base). Thw fluorphore on each strand in the gel is detected and a chromatogram is produced . THe trace is read to determine the sequence of bases in the template. - deoxynucleotides have OH- which allows for chain growth.

things involved in dna replication ^^^

- including the DNA polymerase complex that elongates each strand at the 3′ end, the RNA primase that makes the primer, the DNA polymerase complex that replaces the RNA primer with DNA, and the DNA ligase that joins the DNA fragments in the lagging strand, topoisomerase II (an enzyme that breaks a DNA double helix, roates the end, and seals the break. It works upstream from the replication fork to relieve the stress on the double helix that results from its unwinding at the replication fork. ), single-strand binding protein (A protein that binds single-stranded nucleic acids.), ^^^^enzyme that unwinds DNA= helicase - DNA polmerase extends an RNA primer - topoisomerase II cuts the phospahte backbaone which is eventually released. - single stranded binding proteins binds single stranded DNA regions. They are like spacers. prevent the two templante strands from interactiving with each other - the DNA polymerase complex acts at the site of the growing chain to increase the chain length

recombinant DNA DNA molecules from two (or more) different sources combined into a single molecule. ^^^^^

-Recombinant DNA is the basis for genetically modified organisms (GMOs), which offer both potential benefits and risks -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-stranded DNA at a specific site. ^^^^ - bacteria can make RNA sequence that bind to target DNA for destruction. This is used to protect the bacteria - Key component of how CRISPR (used to edit genomes)works: - Take a single strand of RNA that has a region that is complementary to the region of DNA that we want to change. (this RNA is called a CRISPR RNA) **** - the RNA is then introduced into the organism together with an enzyme called CAS9. The RNA then guides the CAS9 to the target DNA and the DNA is then cleaved.**** - (these are all carried by plasmids) - an exonuclease widens the gap in the target DNA in order for the new sequence to fit. - The editing template is used to repair the gap in the target DNA - The result is an edited DNA with altered sequence.

what makes sure that both strands replicate at the same rate

-The DNA polymerase complexes for each strand stay in contact with each other, with the lagging strand's polymerase releasing and retrieving the lagging strand for the synthesis of each new RNA primer. The positioning of the polymerases is such that both the leading strand and the lagging strand pass through in the same direction, which requires that the lagging strand be looped around - when DNA damage occurs during replication, the rate of synthesis slows down so that the DNA can be repaired. If synthesis of one strand slows down to repair DNA damage, synthesis of the other strand slows down, too. The pairing of the replication complexes is disrupted when the RNA primer of the previous Okazaki fragment is encountered, and then takes place again when a new lagging-strand primer is formed.

The two models for DNA replication: semi conservative replication^^^^ conservative replication

-The mechanism of DNA replication in which each strand of a parental DNA duplex serves as a template for the synthesis of a new daughter strand.^^^^The orignial parental strand is the templant used to make the daughter strand AND is part of the double stranded DNA - which proposes that the original DNA duplex remains intact and the daughter DNA duplex is completely new.

lagging strand ^^^^

-The situation is different for the daughter strand shown at the top - Its 5′ end is pointed toward the replication fork, but the strand cannot grow in that direction. Instead, as the replication fork unwinds, it grows away from the fork and forms a stretch of single-stranded DNA of a few hundred to a few thousand nucleotides, depending on the species - Then, as the parental DNA duplex unwinds further, a new daughter strand is initiated with its 5′ end near the replication fork, and this strand is elongated at the 3′ end as usual. -The result is that the daughter strand shown at the top is actually synthesized in short, discontinuous pieces. As the parental double helix unwinds, a new piece is initiated at intervals, and each new piece is elongated at its 3′ end until it reaches the piece in front of it. The short pieces in the lagging strand are called Okazaki fragments -The presence of leading and lagging strands during DNA replication is a consequence of the antiparallel nature of the two strands in a DNA double helix, and the fact that DNA polymerase can synthesize DNA in only one direction. ^^^ DNA ligase makes the phospister bond which joins the two DNA fragaments.

DNA replication expeirment of Meselson and Stahl**** ^^^^

-found that DNA in fact replicates semiconservatively - HYPOTHESIS DNA replicates in a semiconservative manner, meaning that each new DNA molecule consists of one parental strand and one newly synthesized strand. ALTERNATIVE HYPOTHESIS DNA replicates in a conservative manner, meaning that one DNA molecule consists of two parental strands, and the other consists of two newly synthesized strands. METHOD Meselson and Stahl distinguished parental strands ("old") from newly synthesized strands ("new") using two isotopes of nitrogen atoms. Old strands were labeled with a heavy form of nitrogen with an extra neutron (15N), and new strands were labeled with the normal, lighter form of nitrogen (14N). EXPERIMENT The researchers first grew bacterial cells on medium containing only the heavy 15N form of nitrogen. As the cells grew, 15N was incorporated into the DNA bases, resulting, after several generations, in DNA containing only 15N. They then transferred the cells into medium containing only light 14N nitrogen. After one round of replication in this medium, cell replication was halted. The researchers could not observe the DNA directly, but instead they measured the density of the DNA by spinning it in a high-speed centrifuge in tubes containing a solution of cesium chloride. PREDICTION If DNA replicates conservatively, half of the DNA in the cells should be composed of two heavy parental strands containing15N, and half should be composed of two light daughter strands containing 14N. If DNA replicates semiconservatively, the daughter DNA molecules should each consist of one heavy strand and one light strand. RESULTS When the fully15N-labeled parental DNA was spun, it concentrated in a single heavy band with a density of 1.722 gm/cm3. After one round of replication, the DNA formed a band at a density of 1.715 gm/cm3, which is the density expected of a duplex molecule containing one heavy (15N-labeled) strand and one light (14N-labeled) strand. DNA composed only of 14N would have a density of 1.708 gm/cm3. After two rounds of replication, half the molecules exhibited a density of 1.715 gm/cm3, indicating a duplex molecule with one heavy strand and one light strand, and the other half exhibited a density of 1.708 gm/cm3, indicating a duplex molecule containing two light strands. 1. at the begining of the experiment, both strabds are labeled with heavy nitrogen (15N). The molecules form a band with the density of heavy DNA. 2. After one round of replication in the absence of heavy nitrogen, the parental strand still contains the heavy nitrogen (15N), bu the daughter strand contains light nitogen (14N). The daughter DNA molecules form a band with an intermediate density. 3. After two rounds of replication, half of the duplex DNA molecules have one strand with 15N and one strand with 14N and the other half have both strands with 14N. The daughter DNA molecules therefore form two bands, one intermediate in density and the other light. ^^^^^different density becuase its a hybrid. (parental strand and new daughter strand) why does only one have the 14n- becuase the next round of repication have 14n as a templant. - once the 14N is present after the first round of replication, in the second round of replication there will be two strands with only 14n (light blue)

each cycle of amplification includes three steps PCR repeats the cycle of amplification

1. denaturation: a colution containing double strnaded DNA (the template duplex) is heated to separate the DNA into two individual strands. 2. Annealing: when the solution is cooled the two primers anneal to their complementary sequence on the strands of the template dulplex 3. extentsion: DNA polmerase synthesizes new DNA strands (complmentary to the templat duplex strands) by extending primers in a 5' to 3' direction PCR: 1. the template duplex is often lonerg than the amplified region 2. primers for PCR are typically 20-30 nucleotides in length. their sequences are used for amplification. The primers are chosen to base pair with one or the other strand of the template duplex, with their 3' ends oriented toward each other. The primers are added to te reaction mixutre in great excess to ensure pairing with any complementary sequence 3. each round of amplification dobles the number of molecules that have the same sequence as the template duplex 4. after N cycles of amplification there are 2n copies of the target sequence. When n=30 there are 2^30=10^9 copies. the procedure takes place in a small plastic tube containing a solution that includes four essential components: Template DNA. At least one molecule of double-stranded DNA containing the region to be amplified serves as the template for amplification. DNA polymerase. The enzyme DNA polymerase is used to replicate the DNA. All four deoxynucleoside triphosphates. Deoxynucleoside triphosphates with the bases A, T, G, or C are needed as building blocks for the synthesis of new DNA strands. Two primers. Two short sequences of single-stranded DNA are required for the DNA polymerase to start synthesis. Enough primer is added so that the number of primer DNA molecules is much greater than the number of template DNA molecules.

^^^^^ The rules of DNA replication

1. each strand of the parental double helix serves as a template for synthesis of one of the new daughter strands. So the order of the bases in the parental strands determines the orde in the new strands. A to T and C to G 2. DNA synthesis processds in the 5' to 3' direction 3. the template is read in the 3' to 5' direction 4. the 2 DNA strands in the final duplex are antipararell. The 3' OH of the growing strand attacks the high energy phosphate bond of the incoming nucleotide to inititate the synthesis reaction. - the DNA sequence refers to the identity and order of bases 5' to 3' in one strand. And you can make the other strand but is labebed 3' to 5'

Leading and lagging strand synthesis (DNA replication) ^^^^

1. unwinding of the DNA duplex results in a replication fork. 2. Replication always occurs 5' to 3' direction. The daughter strand on top elongates from left to right, that on the botoom from right to left. 3. Replication of the top strand is disontinuous (fragmeneted) whereas that of the bottom strand is continuous 4. primers are removed and replaced with DNA and the fragements of the discontiuous (lagging) strand are ligated when the meet. ^^^why do we need the primer? - the primer provides a 3' OH so that DNA polymerase can add the first nucleotide of the new DNA strand. Lagging stand synthesis needs many RNA primers. And then after application the primer is removed. -

polymerase chain reaction (PCR) ^^^^

A selective and highly sensitive method for making copies of a piece of DNA, which allows a targeted region of a DNA molecule to be replicated into as many copies as desired. - PCR is both selective and highly sensitive, so it is used to amplify and detect small quantities of nucleic acids, such as HIV in blood-bank supplies, or to study DNA samples as minuscule as those left by a smoker's lips on a cigarette butt dropped at the scene of a crime. The starting sample can be as small as a single molecule of DNA. ^^^ - PCR makes millions of copies of both strands of a target double starnded DNA sequence. - PCR is one the most common technqiues used in molecular biology. Applications include cloning and mutagenesis in research labs, medical diagnostics and forensics - PCR requires a dsDNA target (2 templates). DNA polyerase, deoxynuleotide triphosphates (A,T,G,C) and 2 primers ( you need primers so DNA polymerase knows where to start laying down nucleotides. NEEDS THAT 3' OH!!!!!) - PCR consists of about 25 cycyles of 3 repeating steps in the following order: 1. denaturation (separating the strands. The double stranded DNA is heated (in the lab) to separate it into two indivi strands. This is the first step becuase we need to expose the bases and reveal the template. In nature this process is done by the helicase). 2. annealing ( when cooled two primers anneal to their complementary sequence on each of the strands. This allows the hydrogen bonds to be made between the base pairs. The primers used are not removed as seen in DNA replication in the cell. The primers are also parts of DNA unlike in the cell where the primers are RNA. Primers are essential as they provide the free 3' OH to begin DNA synthesis. They aren't removed becuase they we can make changes in the primers if needed.) 3. extension or elongation. ( DNA polymerase synthesizes new DNA strands (complementary to the template strands) in a 5' to 3' direction.) The polmerase used is one that can survive in high temps. In the cell we get two copies of the double stranded DNA compared to PCR where we get millions of copies. We get millions of copies becuase every new strand made gets copied.

olignucleotides

A short (typically 20 to 30 nucleotides), single-stranded molecule of known sequence produced by chemical synthesis; oligonucleotides are often used as primer sequences in the polymerase chain reaction. -The 3′ end of each primer must be oriented toward the region to be amplified so that, when DNA polymerase extends the primer, it creates a new DNA strand complementary to the targeted region. Because the 3′ ends of the primers both point toward the targeted region, one of the primers pairs with one strand of the template DNA and the other pairs with the other strand of the template DNA.

Suppose Meselson and Stahl had done their experiment the other way around, starting with cells fully labeled with 14N light DNA and then transferring them to medium containing only 15N heavy DNA. What density of DNA molecule would you predict after one and two rounds of replication?

After one round of replication, you would predict only 14N/15N hybrid DNA, which has a density of 1.715 gm/cm3. After two rounds of replication, you would predict half the molecules to be 14N/15N hybrid DNA (density 1.715 gm/cm3) and half to be 15N/15N heavy DNA (density 1.722 gm/cm3).

Mitochondria contain their own double-stranded, circular DNA and replicate on their own. Why don't they suffer the same consequences as our cells in terms of limited division? Because they have high telomerase activity and their DNA is circular and so doesn't shorten in replication. Because mitochondrial DNA is circular, it doesn't shorten when it replicates. Shortening of the DNA is only a problem with nuclear DNA; shortening of mitochondrial DNA isn't catastrophic. Since mitochondrial DNA only contains a few genes, it can last a lot longer. Because they have high telomerase activity.

Because mitochondrial DNA is circular, it doesn't shorten when it replicates.

What procedure could be used after PCR is complete to make certain that the amplification process worked properly and that the correct sequence was amplified? gel electrophoresis DNA sequencing DNA denaturation DNA hybridization None of the answer options is correct.

DNA sequencing

Which of the following statements is true regarding chromosome replication in eukaryotes? During chromosome replication, multiple origins of replication form for all DNA in a cell. Replication bubbles remain distinct, isolated structures during chromosome replication and never fuse with one another. In humans, it takes several days to replicate an entire chromosome. Replication begins at one end of the chromosome and proceeds until it reaches the opposite end. None of the answer options is correct.

During chromosome replication, multiple origins of replication form for all DNA in a cell.

Imagine that a doctor is culturing cells from a malignant melanoma and from a normal skin sample. How would you expect these two cell populations to differ? Normal skin cells would have telomeres that do not shorten during successive rounds of replication. Normal skin cells would have active telomerases that constantly replenish and lengthen telomeres. Malignant melanoma cells would have active telomerases that constantly replenish and lengthen telomeres. Malignant melanoma cells would have telomeres that do not shorten during successive rounds of replication. Malignant melanoma cells would have inactive telomerases and so their telomeres would shorten during successive rounds of replication.

Malignant melanoma cells would have active telomerases that constantly replenish and lengthen telomeres.

PCR creates new DNA fragments in a cycle of three steps,

The first step, denaturation, involves 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. The second step, 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 (rather than two strands of the template duplex coming back together). In the final step, extension, the solution is heated to the optimal temperature for DNA polymerase and the polymerase elongates (or "extends") each primer with the deoxynucleoside triphosphates. -In each cycle, the number of copies of the targeted fragment is doubled. The first round of PCR amplifies the targeted region into 2 copies, the next into 4, the next into 8, then 16, 32, 64, 128, 256, 512, 1024, and so forth. By the third round of amplification, the process begins to produce molecules that are only as long as the region of the template duplex flanked by the sequences complementary to the primers. After several more rounds of replication the majority of molecules are of this type. The doubling in the number of amplified fragments in each cycle justifies the term "chain reaction."

replication fork

The site where the parental DNA strands separate as the DNA duplex unwinds. - this is untrue because The strand that terminates in the 5′ phosphate cannot grow in the direction of the replication fork because new DNA strands can grow only by the addition of successive nucleotides to the 3′end. That is, DNA always grows in the 5′-to-3′ direction.

On which strand are new nucleotides being added in the same direction as the replication fork is opening? Question 3 choices Choice A., the leading strand Choice B., the lagging strand Choice C., On both strands, DNA synthesis is proceeding in the same direction as the replication fork.

choice A. because the new nucleotides can be added only at the 3' end of a growing DNA strand, one daughter strand is elongated in a direction toward the replication fork.

RNA primase lays down an RNA primer to start DNA replication. If a mutation occurs that blocks the function of RNA primase, which synthesis of which strand is affected, and why? Question 6 choices Choice A., The leading strand is affected because it leads DNA synthesis. Choice B., The lagging strand is affected because it requires an RNA primer for each Okazaki fragment produced. Choice C., Both strands are affected because all DNA polymerase can only add new nucleotides to a 3′-OH present at the end of the RNA primer.

choice C. both strand use an RNA primer to start DNA replication

What is the process of "proofreading" by DNA polymerase? Question 5 choices Choice A., re-replicating a template of DNA for a second time Choice B., removing an improperly paired base and replacing it with the proper one Choice C., changing the DNA sequence to produce improved protein structures Choice D., removing introns from the sequence and joining exons to each other

choice b

When DNA polymerase progresses during DNA replication, how is the correct new nucleotide selected to be next? Question 1 choices Choice A., DNA polymerase chooses which nucleotides can form phosphodiester bonds. Choice B., DNA polymerase chooses different nucleotides for the leading and lagging strands. Choice C., DNA polymerase chooses nucleotides that undergo proper base pairing with the corresponding base on the template strand. Choice D., DNA polymerase chooses nucleotides that can extend from the RNA primer. Submit

choice c

Which of the following does not occur after an incorrect base is removed by DNA polymerase? Question 4 choices Choice A., Polymerase activity is restored. Choice B., New nucleotides are incorporated into the growing daughter strand. Choice C., The template and daughter strands are separated by helicase activity. Choice D., The correct nucleotides are moved into place and attached. Submit

choice c

How frequently is an improper base incorporated into the growing daughter strand? Question 2 choices Choice A., frequently Choice B., about half the time Choice C., each time there is a purine rather than a pyrimidine on the template strand Choice D., very rarely

choice d

^^^^plasmid

circuliar double stranded DNA. - natually exists in bacteria - hold genes that are useful to the bacteria (such as antibiotic resistance) - AKA vector -

Telomerase activity

differs from one cell type to the next. It is fully active in germ cells, which produce sperm or eggs, and also in stem cells, which are undifferentiated cells that can undergo an unlimited number of mitotic divisions and can differentiate into any of a large number of specialized cell types. -Stem cells are also found in some tissues of the body after embryonic development, where they replenish cells that have a high rate of turnover, such as blood and intestinal cells, and play a role in tissue repair. In contrast to the high activity of telomerase in germ cells and stem cells, telomerase is almost inactive in most cells in the adult body. In these cells, the telomeres are actually shortened by about 100 base pairs in each mitotic division. Telomere shortening limits the number of mitotic divisions that the cells can undergo because human cells stop dividing when their chromosomes have telomeres with fewer than about 100 copies of the telomere repeat. Adult somatic cells can therefore undergo only about 50 mitotic divisions until the telomeres are so short that the cells stop dividing. Many biologists believe that the limit on the number of cell divisions explains in part why our tissues become less youthful and wounds heal more slowly with age. - observed in cancer cells, in which telomerase is reactivated and helps support the uncontrolled growth and division of abnormal cells. ^^^^ - we need to replicate the entire chromosome, so the use of telomeres at the lagging strand. - GGATT - the ends of eukarotic (linear) chromosoes are capped wit telomeres. Telomerase contains a RNA template that binds to the 3' overhang of the template strand, so the telomere (GGATT in humans) can be added. - as we grow older the telemeres arent being put in. we loose the telemeres as we grow older - if we arent making anymore telemeres it tells the cell= no more cell division. - all telemeres have the same sequence and there always needs to be telemeres put in when replicating DNA. - the telemeres are placed on both strands. - telemere shortening leads to a decrease in mitosis, Telomerase activity is high in germ cells and stem cells (undifferentiated cells) but low in the majority od adult somatic cells. if telomerase cant restore telemeres the number of cell division in these somatic cells is small.

RNA primer

each new strand of DNA must begin with a short strech of RNA. - the primer is needed because the DNA polymerase complex cannot begin a new strand on its own; it can only elongate the end of an existing piece of DNA or RNA - The primer is made by an RNA polymerase called RNA primase, which synthesizes a short piece of RNA complementary to the DNA template and does not require a primer. Once the primer has been synthesized, the DNA polymerase takes over and elongates the primer, adding successive DNA nucleotides to the 3′ end of the growing strand. -Because the DNA polymerase complex extends an RNA primer, all new DNA strands have a short stretch of RNA at their 5′ end. For the lagging strand, there are many such primers, one for each of the discontinuous fragments of newly synthesized DNA. As each of these fragments is elongated by DNA polymerase, it grows toward the primer of the fragment in front of it. When the growing fragment comes into contact with the primer, a different DNA polymerase complex takes over, removing the RNA primer and extending the growing fragment with DNA nucleotides to fill the space left by removal of the RNA primer. When the replacement is completed, the adjacent fragments are joined, or ligated, by an enzyme called DNA ligase

Telomerase activity in stem cells is very low. true false

false