Section 1: Genes and Genome

Question 1

Draw out a 3 nucleotide polymer of both DNA and RNA

Answer 1

Check answer online or in notes

Question 2

What is the central dogma of biology?

Answer 2

DNA can replicate itself and can be transcribed into RNA. RNA can then be translated into proteins, and the proteins carry out specific functions. RNA can also be reverse transcribed back into DNA.
In one sentence: “Genetic information can be transmitted from nucleic acids to proteins, but never the other way around”

Question 3

Explain the first major experiment that was done to isolate and identify the genetic material

Answer 3

• Controversy was whether proteins or DNA were the genetic material, and people generally though proteins were, simply because there were more amino acids than nucleotides
• Griffith discovered the “transforming principle” of genetic material
• He was working with peumacocans bacteria, which comes in a smooth variety and rough variety. The smooth variety were virulent and their smoothness was this “coat” they had that protected themselves from the host’s immune system. The rough didn’t have this coat, so were non-virulent.
  The experiment consisted of injecting the bacteria into mice in the following ways:
  1. Injected smooth bacteria into mice —> mice died
  2. Injected rough bacteria into mice —> mice lived
  3. Heat killed smooth bacteria and injected into mice —-> mice lived
  4. Heat killed smooth bacteria, mixed with rough bacteria, and injected into mice —–> mice died
• This indicated there was something in the heat killed smooth bacteria that the rough bacteria could take up
• It wasn’t just that the rough bacteria were “borrowing” the smooth bacteria’s coats, b/c colonies of smooth bacteria were obtained from this mixture, and there aren’t enough “coats” to go around.

Importance: could isolate each chemical compound of the heat killed smooth bacteria, mix with rough bacteria and grow colonies to see which gave smooth bacteria colonies. The mixture that did contains the genetic material.

• Scientists did this and found that DNA was compound that gave smooth bacterial cultures.
• People still weren’t really convinced that DNA was genetic material, however

Question 4

Explain the second major experiment that was done to isolate and identify the genetic material

Answer 4

• Phages consist of only a protein coat and DNA.
• They work by injecting their host cell with their genetic material so the host cell makes more viruses until the cell eventually bursts, releasing the phages
• Scientists labeled a set of phages with the radioactive label S35, which would label the proteins, and another set of phages with P32, which would label the DNA.
• They allowed the phages to inject into the bacteria, removed the phages, and then looked at the cell to see the radioactivity
• The P32 radioactivity was seen inside the cell, confirming DNA was the genetic material
• https://en.wikipedia.org/wiki/Hershey–Chase_experiment

Question 5

Watson and Crick and the 3-D DNA structure

Answer 5

• Used two main pieces of data: the x-ray diffraction data of DNA obtained by Franklin, which they recognized made a double helical structure, and Chargoff’s rule that [A]=[T] and [C]=[G], or essentially that # purines = # pyrimidines.
  Their model included the following:
• C binds to G via 3 hydrogen bonds, and A binds to T via 2
• DNA exists in a double helix
• The two strands of DNA are anti-parallel
• The bases are inside, facing each other to hydrogen bond together, and the sugar-phosphate structure creates the backbone

Question 6

Important Implication of DNA being a double helix

Answer 6

• Double helical structure with two anti-parallel strands provided a mechanism of replication, in which one strand could be used as a template to make the new strand, so each new dsDNA had an old and new strand. This idea still needed to be proven, however
• this would also serve as a way for mutations to be passed down since the template strand is “read” to make the complementary strand

Question 7

Explain the experiment to determine the mechanism of DNA replication

Answer 7

• Was an experiment to test which replication mechanism DNA utilizes
• Took 15-N labeled DNA strand and put it in a medium that contained 14-N nucleotides and allowed replication occur
• Afterwards they would centrifuge it and see what bands would appear
• Three different bands could appear: Heavy DNA band which was all 15-N, medium DNA band which was half 15-N, half 14-N, or a light band which was all 14-N
• If conservative, they would expect to see one heavy band and one light band after one round
• If semiconservative, they would expect to see one medium band after one round
• After one round of replication they had one band of medium DNA
• After another round they had one band of medium DNA and one band of light DNA
• This was evident of the semi-conservative mechanism

Question 8

Necessary Ingredients for in vitro DNA replication

Answer 8

• DNA polymerase
• Single stranded template DNA (or dsDNA and a helicase?)
• dNTPs
• Primers

Question 9

In what direction is DNA replication carried out in, and why?

Answer 9

• In the 5’ to 3’ direction of the strand being made, so in other words, DNA polymerase adds onto the 3’ end of the new strand
• Replication is carried out in this direction due to evolutionary favorability due to the phosphate group being on the 5’ end and the hydroxyl group on the 3’ end
• The energy stored in the triphosphate group is used to drive the formation of the phosphodiester bond when two nucleotides are added together when the hydroxyl group on one attacks the triphosphate of the other. The triphosphate group is relatively unstable in the sense that it can be hydrolyzed relatively easily, even by things that aren’t the hydroxyl group of another nucleotide. If we elongated from the 5’ end, we would need to use the triphosphate of the primer, and then of each subsequent nucleotide. The triphosphate may get hydrolyzed before the next nucleotide can come in, however, which would cut off replication. If we add to the 3’ end, we have to worry about our dNTPs getting hydrolyzed. This isn’t as big of a deal, however, since there are lots of them, and we can use any one of them (as long as it is the correct base, that is) to add on to our growing chain. This eliminates the concern of replication getting cut off too soon due to unwanted hydrolysis.

Question 10

Why Doesn’t RNA follow Chargaff’s Rule?

Answer 10

• Mainly because it is usually single stranded
• It has the capability to form bases pairs from hydrogen bonding, but it typically doesn’t, and usually exists as a single strand
• While it is true that RNA has different bases than DNA, this is not a reason why it doesn’t follow Chargaff’s rule.

Question 11

Molecular Cloning

Answer 11

Online Def: The process of assembling recombinant DNA molecules and to direct their replication within the host genome
The five steps are
1. Cutting your desired piece of DNA from the genomic or viral DNA
2. Paste your piece of DNA into a vector
3. Transform the vector into cells
4. Select Transformations
5. Check Plasmid on a gel

Question 12

First step of Molecular Cloning and Isolating a Gene of Interest

Answer 12

• Want to cut the genomic DNA with restriction enzymes around the sequence of interest. We of course need restriction enzyme sequences in our DNA for this to work
• Mix our genomic DNA in with our restriction enzyme to generate the cuts
• We’ve now separated out sequence of interest from our genomic DNA, but this isn’t good enough because
  1. We only have a small quantity; we need more so we can study it
  2. This piece is non-functional in the cell. Bacterial cells don’t know what to do with a short piece of DNA b/c it isn’t circular like a plasmid like their genomic DNA, so they degrade it
• We can solve both problems by inserting our gene of interest into a vector

Question 13

Restriction enzymes

Answer 13

• Enzymes that recognize specific palindromic sequences and cut
• Is an endonuclease
• Breaks the phosphodiester bond that was between those two nucleotides, so the nucleotide that had that phosphate group keeps it and the other regenerates its hydroxyl group
• Can cut in one of two different ways: blunt or sticky
• Sticky ends: result when the restriction enzyme makes staggered cuts, having 2-4 nucleotides of each individual strand “hang off”. This allows the strands to hydrogen bond to things more easily
  Blunt ends: When the restriction enzyme cuts right down the middle, cleaving both strands at opposing phosphodiester bonds so there is no overhang

Question 14

Single strand overhang

Answer 14

• Results when a restriction enzyme makes staggered cuts

- Also called a sticky end

Question 15

Vector

Answer 15

• Plasmid or viral DNA that can replicate in a desired organism

Question 16

Second step of Molecular Cloning

Answer 16

• Inserting the gene of interest into a vector to be replicated
• Need to get a vector that has the same restriction sites as our gene of interest (in the correct orientation, too) and cut it with restriction enzymes as well
• This generates ends that are complementary to our gene of interest’s ends, so they can form hydrogen bonds with one another
• We then mix in our cut vector with our gene of interest and add ligase to ligate them together by forming the sugar-phosphate bonds. Ligase needs ATP, so we need to supply it ATP as well

Question 17

Essential components of a plasmid

Answer 17

• Correct restriction sites
• Origin of replication- where the cell recognizes to start replication
• Selectable marker- something that allows us to distinguish cells that took up our plasmid from cells that didn’t; is typically an antibiotic resistance marker

Question 18

Third Step of Molecular Cloning

Answer 18

• Transforming your vector into bacteria
• The bacteria will suck in outside DNA in a process called “transforming”
• Each bacterial cell can only take in one piece of DNA
• Transformation efficiency is rather low, so to be able to distinguish between the bacteria that took up our plasmid and the bacteria that didn’t we can plate it on a plate with our antibiotic marker, and only bacteria with our plasmid which has an antibiotic resistance gene will be able to form colonies

Question 19

If our origin of replication is missing in our plasmid, will we still get colonies?`

Answer 19

• No, b/c without the origin of replication, replication can’t occur. The cell will survive b/c it has an antibiotic marker, but it won’t form a colony

Question 20

Step 4 of molecular cloning

Answer 20

• Picking colonies and growing up the bacteria to get more of our bacteria, and thus our gene of interest

Question 21

Step 5 of molecular cloning

Answer 21

• Running a gel to see if our plasmid is
• Do so by isolating the DNA form the colonies we grew up and then cutting them with our restriction enzymes again in different combinations (ex. cutting with just EcoRI, then cutting with EcoRI and Bam HI)
• Then run it on the gel and see what bands we see; if we don’t see the bands we expect to see, then our plasmid most likely had a problem and/or our gene of interest isn’t present

Question 22

Molecular Cloning Before Genome Sequencing

What problem made this more difficult?

Answer 22

• We don’t know where our gene is in our genomic DNA
• Ex. Used in a mutant in ARG1 in a yeast cell preventing it from making arginine
• We treat our genomic DNA with various restriction enzymes and hope they don’t cut through our gene
• Next we ligate them into plasmids and transform bacterial cells with our plasmids
• We then pick multiple colonies and extract the plasmid from them. Of course, we don’t know which has our gene of interest, if any
• We can then transform the vector into our organism of interest that has a mutant in that gene. The plasmid that does NOT produce a wild type phenotype is the plasmid with our gene of interest.
• For example, if we take the genome from a yeast cell with a non mutated ARG1 and then transform the plasmid with the non mutated ARG1 into the mutated yeast and it produces ARG1, we know that that gene in our plasmid is our gene of interest. If we don’t produce ARG1 after transformation, that means the plasmid contained some random piece of our genomic DNA that we don’t care about.
  This process of looking for the plasmid with our gene of interest by seeing if it will produce a certain phenotype in an organism with that gene mutated is called screening.
• We can then determine the sequence of our gene of interest using Sanger sequencing

Question 23

Screening

Answer 23

Online Def: an experimental technique used to identify and select for individuals who possess a phenotype of interest in a mutagenized population

Question 24

Sanger sequencing

Answer 24

• A method for determining the sequence of nucleotides in DNA through the controlled termination of replication
• We take our DNA to be sequenced and put it in with our DNA polymerase I, labeled dATP, TTP, dCTP and dGTP as well as a dideoxy analog of one of these dNTPs
• We only want a small amount of our dideoxy analog present, b/c if we have too much it will outcompete the other dNTP and we will get termination of replication at the first place that nucleotide needs to be incorporated
• We essentially do this a bunch of times and let it run, and we will get a shit ton of fragments with different termination spots, which will tell us where all of that nucleotide is located in the DNA.
• Then we can redo this for all the other nucleotides to see where they are
• We can then run a gel and see how far each band travels to determine the DNA sequence by running each ddNTP experiment next to each other in the gel
• When reading , the band closest to the + side of your gel is your 5’ end of the DNA sequence
• You have to denature your gel before loading, or run it on a denaturing gel
• More efficient method:
• Attach fluorescent tags to each dideoxy analog so we can see where each nucleotide is
• This allows us to run all four dideoxy analogs at once if we use a different tag on each dideoxy analog so we can save time
• We then obtain a chart that shows us the location of each tag in the sequence, and we typically only get one tag for each position, which tells us that that is the nucleotide present there
• The sizes of the peaks don’t matter, all they are telling us is that we have more fragments that are of that length
• We determine what the order is by running a column so the smaller fragments come out first and larger things come out later to tell us where they are in the sequence

Question 25

Why can’t we use Sanger Sequencing for Genome Sequencing?

Answer 25

• Sanger sequencing can only sequence things that are about 1000 bp, b/c when you run a gel, anything bigger than this won’t separate out if they’re only 1 bp apart, or a few bp apart
• One way to get around this limit, however, is to cut the genomic DNA with restriction enzymes, ligate the pieces into plasmids, transform into bacteria, pick colonies, and then isolate the DNA and sequence each fragment
• There should be some overlap in sequences from using restriction enzymes, so computational tools can be used to align the pieces together to create the entire genome

Question 26

PCR

Answer 26

• Polymerase Chain Reaction
  -A method of copying a specific sequence of DNA over and over again to get multiple copies of it
• You have your DNA strands and you make two different primers for the 5’ end and the 3’ end (5’ end on the opposing strand) of the portion of DNA you want to replicate (typically a specific gene) so it anneals to the 3’ end of the both strands of the template DNA. These primers are complementary to the 3’ flanking regions on each strand of DNA
  -Add heat (95 °C) to denature/separate the double stranded DNA and add in excess primers (Melting)
• We are using high temperatures b/c we are using Taq polymerase which is from an orgamism that lives in hot environments so Taq is used to these environmets
• Cool down to 54 °C (about; it depends on the GC content of your primers) to anneal your primers (Annealing)
• Heat up to 72 °C so taq polymerase does its thing and elongates the DNA chains (Elongation)
• We get two truncated strands after the first round of PCR
• After second round, we start getting our target strands; we have our target DNA sequence attached to each truncated strand ( we have ssDNA of the target DNA sequence)
• We don’t have a copy of our actual DNA sequence that we want until the third round, in which we will have produced
• To calculate the number of target strands you’ll have, take the cycle number you are on, subtract one from it, and take 2 to the power of that number.
  Ex: 4th round
  2^(4-1) = 8 target sequences
• Note that these are single stranded copies, this is NOT counting them as double stranded copies
• We can then run our PCR product on a gel and isolate out our fragments
  Remember: Primers typically ARE NOT complementary to each other
  Remember: We also need Primers to anneal at approximately the same temperature

Question 27

Designing Primers

Answer 27

• When adding restriction sites onto primers, they need to be added on the 5’ end OF THE PRIMER. This is b/c the 3’ end of the newly synthesized DNA chain is what we are elongating off of/is the growing end, so we need it to be free.
• The restriction sites don’t need to (and won’t) be complementary to the sequence; they will get incorporated into the sequence after a couple rounds of PCR

Question 28

Experiment to determine if DNA replication is uni-directional or bi-directional

Answer 28

• B. subtilis cells were synced so they were all in the DNA replication stage?
• The cells were treated with two rounds of radioactivity
• First, the cells were treated with a pulse of a low concentration of radioactivity, then they were treated with a pulse of a high concentration of radioactivity. The idea was to be able to track the incorporation of dNTPs
• The result was the dNTPs in the middle of the replication bubble were slightly radioactive, and the dNTPs at the ends of the replication bubble were more radioactive, indicating that the replication bubble grows in both ways and that DNA replication is bi-directional

Question 29

Origin of replication

Answer 29

• This is a sequence that the cell recognizes as where to initiate replication; helices then comes in at this sequence and starts unwinding DNA, and after the DNA is unwound enough and opens up, primase makes the RNA primers

Question 30

Replication Fork

Answer 30

• Where the replication bubble is expanding at the moment via the helicase
• The helicases are always located where the replication forks are

Question 31

Helicase

Answer 31

• They “unzip” the double stranded DNA and open it up so replication can occur
• It requires ATP to function

Question 32

Primase

Answer 32

• Makes primers for a polymerase to bind to to initiate replication

Question 33

DNA polymerase

Answer 33

• Synthesizes DNA by adding nucleotides onto the 3’ end of the growing DNA strand
• Also has endonuclease activity b/c it is able to remove a single nucleotide from the 3’ end as well in a proofreading mechanism
• It typically does this for mismatched nucleotides because it is more energetically favorable for it to, but it also occasionally does this on nucleotides that are supposed to be there.

Question 34

Leading strand vs Lagging strand

Answer 34

• The leading strand is the strand where only one primer will be needed and we can just elongate off that because the replication bubble is growing on the 3’ end of the primer, meaning it is growing in the same direction the polymerase is elongating in
• The lagging strand is the strand that will have multiple Okasaki fragments that will need to be ligated together
• The lagging strand will need multiple primers, which is how the Okasaki fragments are formed
• We will need multiple primers b/c the replication bubble is growing on the 5’ end, the opposite end that the DNA elongation is happening
• Both the top and the bottom strands of dsDNA will have a lagging strand and a leading strand, just when we are shown a picture of only one half of the replication bubble and one replication fork, it appears that only one is on top and only one is on bottom
• The lagging strand needs a few extra enzymes
• It needs RNase to remove the RNA primers before we can ligate together, it needs DNA polymerase to fill in the gaps made by the removal of the RNA primers, and it needs ligase to ligate the Okasaki fragments together

Question 35

Topoisomerase

Answer 35

• Cuts the strands of DNA surrounding the replication bubble to release the tension that has built up due to the opening of the replication bubble, and then connects the strands back together, but uncoiled

Question 36

Nuclease

Answer 36

• RNA Nuclease (RNase) is used to remove the RNA primers of the lagging strand in DNA replication
• Is an enzyme that cleaves DNA fragments

Question 37

DNA Replication Overview

Answer 37

• First, helicase come in and binds to the origin of replication?, unwinding the DNA and separating the two strands to form a replication bubble
• Primase then adds the RNA primers to both strands of DNA at the ends of the replication bubble
• DNA polymerase then binds to the primers and elongates them to create new DNA strands in the 5’ to 3’ direction of the new strand being synthesized
• The helicase is still unwinding and enlarging the replication bubble this whole time, and then primase keeps adding primers to the lagging strand and DNA polymerase synthesizes them until it reaches the end of the strand in front
• When replication is done, RNA Nuclease (RNase) comes in and removes the RNA primers in the lagging strands
• DNA polymerase then needs to fill in the gaps created by the removal of the RNA primer
• Ligase then ligates the fragments together to create a single stand
• While helicase is unwinding, topoisomerase is slightly ahead of it, cutting the dsDNA that has become super entangled and tense due to the enlarging of the replication bubble and then connecting the strands back together, but now untangled

Question 38

Ligase

Answer 38

• Ligates pieces of DNA together by attaching the hydroxyl group of the 3’ end of one to the phosphate group of the 5’ end of the other. It requires ATP

Question 39

Mismatch excision repair

Answer 39

• A method of fixing replication errors
• The wrong base is recognized in the daughter strand
• DNA helicase opens up the DNA, and endonuclease cuts, and then exonuclease removes said mismatch in the daughter strand
• The gap is then repaired by DNA polymerase and DNA ligase
• The cell knows which strand is the daughter strand and which is the parent strand b/c the parent strand is methylated

Question 40

General Strategy of DNA repair in single strand damage

Answer 40

1. Damage/error recognized
2. Damaged/erroneous DNA strand or nucleotide excised (nucleases)
3. Gap filled with DNA polymerase and DNA ligase

Question 41

Homologous recombination

Answer 41

• One mechanism to repair a double stranded DNA break
• The undamaged sister chromatid serves as a template
• This only works if we are in the stage of the cell cycle after replication? and sister chromatids exist

Question 42

End-joining

Answer 42

• A mechanism to “repair” a double stranded DNA break
• Two separated ends are joined, or two different broke ends are joined
• This results isa mutation, which the cell hopefully will recognize so it can commit suicide