Section 1: Genes and Genome Flashcards
Draw out a 3 nucleotide polymer of both DNA and RNA
Check answer online or in notes
What is the central dogma of biology?
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”
Explain the first major experiment that was done to isolate and identify the genetic material
- 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
Explain the second major experiment that was done to isolate and identify the genetic material
- 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
Watson and Crick and the 3-D DNA structure
- 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
Important Implication of DNA being a double helix
- 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
Explain the experiment to determine the mechanism of DNA replication
- 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
Necessary Ingredients for in vitro DNA replication
- DNA polymerase
- Single stranded template DNA (or dsDNA and a helicase?)
- dNTPs
- Primers
In what direction is DNA replication carried out in, and why?
- 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.
Why Doesn’t RNA follow Chargaff’s Rule?
- 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.
Molecular Cloning
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
First step of Molecular Cloning and Isolating a Gene of Interest
- 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
Restriction enzymes
- 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
Single strand overhang
- Results when a restriction enzyme makes staggered cuts
- Also called a sticky end
Vector
- Plasmid or viral DNA that can replicate in a desired organism
Second step of Molecular Cloning
- 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