Chapter 6.2 Flashcards
What are the 7 basic steps to DNA replication?
- Helicase unwinds the DNA
- Single Stranded Binding Proteins protect the free DNA
- Primase synthesizes RNA primers to attract DNA polymerase
- DNA Polymerase binds and synthesizes DNA
- RNA primers are removed
- DNA Ligase joins Okazaki Fragments
- Topoisomerase reduces tension in the advancing coil
How many primers does the leading strand require?
1
How many primers does the lagging strand require?
many
What is Primase?
Primase is an enzyme that lays down RNA primers for DNA polymerase to bind to.
How are RNA primers removed and replaced in Prokaryotes versus Eukaryotes?
RNA primers are removed and replaced in prokaryotes by DNA polymerase I.
RNA primers are removed in Eukaryotes by RNase H. They are replaced by DNA Polymerase delta
What is DNA ligase?
DNA ligase is a molecule that can join together fragments of unconnected DNA. DNA ligase joins okazaki fragments together!
How is the DNA polymerase in prokaryotes and eukaryotes different?
In prokaryotes, DNA is replicated by DNA polymerase III.
In Eukaryotes, DNA is replicated by DNA polymerase alpha, delta, and epsilon.
What are the 5 classic DNA polymerases in Eukaryotic cells?
Alpha, Beta, Gamma, Delta, and Epsilon.
What is the combined role of DNA pol Alpha, Delta, and Epsilon?
Alpha (pol α-DNA primase complex) lays down primer and starts replication and is then replaced by delta and epsilon
What is the role of DNA polymerase Gamma?
It replicates mitochondria DNA
What eukaryotic dna pols are involved in repair ?
DNA polymerases β and ε are important to the process of DNA repair.
What is the combined role of DNA pol delta and epsilon?
DNA polymerases δ (delta) and ε (epsilon) are assisted by the PCNA protein, which assembles into a trimer to form the sliding clamp.
The clamp helps to strengthen the interaction between these DNA polymerases and the template strand.
What are the classic prokaryotic DNA enzymes?
- DNA pol I
- DNA Pol II
- DNA Pol III
What are oncogenes?
Oncogenes are pieces of DNA that, if damaged (mutated) promote cell division and cancer.
What type of genetic info is more likely to be an oncogene?
Most of the oncogenes are genes that have to do with cell-cycle and division timing. Your eye color gene, on the other hand, probably wont give you cancer if it mutates.
What are antioncogenes?
Antioncogenes are genes that stop and slow tumor progression (Also called tumor suppressor genes, p53 and Rb). This can be because they prevent mutations to DNA in the first place, fix them after they happen, or kill cancer cells. When antioncogenes are mutated, they lose their anti-tumor properties.
What are the two main ways DNA polymerase prevents errors in replication?
- Proofreading
- Mismatch Repair
Describe how DNA polymerase proof-reads bases.
Part of the DNA polymerase enzme is specifically designed to proofread. When complementary strands have incorrectly paired bases the hydrogen bonds are unstable. The proofreading section of DNA polymerase detects the unstable hydrogen bond and removes the incorrect base. All prokaryotic polymerases and DNA epsilon and delta have 3’-5’ exonuclease activity
Describe Mismatch Repair
In the G2 phase of the cell cycle, special enzymes will check the DNA for errors missed by DNA pol during S phase and correct them. Uses genes MSH2 and MLH1
In the G2 phase of the cell cycle, special enzymes will check the DNA for errors missed by DNA pol during S phase and correct them. Uses genes MSH2 and MLH1
G1 and G2
Describe Nucleotide Excision Repair?
In nucleotide excision repair, a section of a damaged DNA strand is completely cut out, phosphodiester back bone included, by excision endonuclease. Next, DNA polymerase fills in the gap, using the undamaged strands as a template. The fixed strand is sealed back into place with DNA ligase. Used to repair Thymine dimers and other helix deforming lesions (chemical carcinogens or UV make bulky lesions)
Describe Base Excision Repair?
In base excision repair, a damaged base is detected and removed by a glyosylaze enzyme, leaving behind an empty backbone called an AP site (apurinic/apyramidic, or abasic site). AP endonuclease recognizes the empty backbone and cuts it out. DNA polymerase can then synthesize a complementary strand using the undamaged strand. DNA ligase seals the repaired strand back into place. Used to repair non deforming mismatches like cytosine deamination (cytosine loses amino group and becomes uracil) (oxidative damage, AP sites, and alkylation)
What are restriction enzymes?
Restriction enzymes (also known as restriction endonucleases, a sub class of endonucleases) are enzymes that recognize palindromic double stranded DNA sequences and cut them. These enzymes are isolated from bacteria and are used to cut up DNA strands in the laboratory.
A palindromic sequence of double stranded DNA is a strand of DNA that has the same 5’ to 3’ sequence on both strands, they’re just going in opposite directions.
What is a vector in the context of DNA Biotechnology?
A vector is usually a bacterial or viral plasmid that can be transferred to a host bacterium.
What is a recombinant vector in the context of DNA Biotechnology?
A recombinant vector is a vector that has been modified with new DNA.
How do you make a recombinant vector?
First, you use a specific restriction endonuclease to cut a certain piece of the vector’s DNA out.
Then, you use the same specific restriction endonuclease to cut a piece of the DNA you are interested in out.
Because you used the same endonuclease, the foreign DNA will be able to fit the hole made in the vector DNA.
Finally, you just mix the foreign DNA fragment with the vector, mix them in annealing conditions and treat with DNA ligase to lock them into place.
What is the point of making a recombinant plasmid vector?
When you put a little piece of DNA you are interested in into a recombinant vector, you can insert it into a bacterium.
The bacterium will grow, and in so doing will replicate its DNA, including the recombinant vector. To find which bacterias have recombinant vector, you can encode antibiotic resistence on vector and then grow bacteria on antibiotic plate.
By letting big colonies of bacteria grow with your DNA you can generate large quantities of it.
Once you feel like you have enough bacteria, you can either kill the bacteria and re-treat them with the endonucleases to collect all the DNA the bacteria made for you or you can make them express the gene so you can collect all the protein that gets made from that DNA. (this is how we make insulin, we grow it in bacteria!)
What are DNA libraries?
DNA libraries are large collections of known DNA sequences that are cloned into vectors for use in future studies. DNA libraries can be Genomic or Complementary
What is the difference between a Genomic Library and a Complementary (expression) Library? *** table
A genomic DNA library contains large fragments of DNA that are digested out of chromosomal DNA itself, which means these strands of DNA include both coding (exon) and noncoding (intron) regions. This method is preferred for sequencing the entirety of an organism’s genome. Cloned genes may be split into multiple vectors, so you can’t do anything with specifc genes. Uses restriction endonuclease and ligase
A complementary DNA library (aka expression library) is constructed by reverse-transcribing processed mRNA, which means these strands only contain the exons of the DNA from which they came (no introns or promoter/enhancer sequences). Each clone containes the complete gene. This method is generally preferred for sequencing specific genes to identify their function. It is also the only way to make transgenic animals and express genes in animals from which the DNA did not originate (producing insulin). Uses reverse transcriptase and ligase
What is Hybrdiziation? (in terms of DNA)
Hybridization is the process of bringing two complementary base pair sequences together. This can be DNA-DNA recognition or DNA-RNA recognition. Used in PCR and southern blotting
Describe PCR
PCR is an a process that can produce millions of copies of a DNA sequence without using bacteria.
During PCR, DNA of interest is placed in a solution of nucleotides, denatured (heated up), replicated with custom made primers (you usually have to buy these for a project, 40-60 percent GC optimal), and then cooled back down. Each round of PCR doubles the amount of DNA, so it only takes a few rounds before you have tons of DNA. Uses Taq polymerase because it functions at high temps
Describe Gel electrophoresis for DNA
DNA is negatively charged, and therefore will migrate towards the positive anode of the electrolytic cell. Larger DNA will migrate slower, just like in protein electrophoresis. Agarose is the preferred gel for DNA gel electrophoresis.
What is a southern blot?
A southern blot is used to detect the presence and quantity of various DNA strands in a sample. In this method, DNA is cut by restriction enzymes in specific ways and then separated by gel electrophoresis. The separated DNA fragments are then transferred to a membrane where they are exposed to a probe. The probe is radioactive single stranded DNA sequence that is visible under x-ray.
If the sequence in the DNA and the probe match up, they will hybridize, and that band of DNA on the membrane will be visible.
How does DNA sequencing work?
DNA sequencing uses dideoxyribonucleotides (ddATP, ddGTP, etc.) to determine the sequence of a segment of DNA.
Dideoxyribonucleotides don’t have a hydroxyl at Carbon 3, and will end DNA replication when they are incorporated into the DNA. Eventually the sample will be composed of a bunch of fragments that end in the ddnucleotide of interest. Then, using gel electrophoresis, you can see how long each fragment was when the ddnucleotide was incorporated. The ddnucleotide is easily read (fluorescent?), and so you can use this with the size seperation to determine nucleotide order
What is gene therapy?
Gene therapy is a therapy for genetic diseases that involves replacing mutated or inaccurate copies of genes in humans with normal copies.
Modified Viruses are usually used to deliver the working gene to the host’s DNA. Small chance that they activate oncogene and give you cancer
What are transgenic mice?
Transgenic mice are mice that had a cloned gene (transgene) insterted into their zygote or some other embryonic stem cell, meaning they have the gene in all of their adult cells. Transgenes usually carry genetic diseases so that we can study the affects of the disease from early embryonic develoment through adulthood.
What is a knockout mouse?
A knockout mouse is a mouse that had one of its genes removed as a zygote, meaning it lacks the gene completely as an adult. Knockout mice are used to study the importance and effects of different genes.
The more GC pairs in a DNA strand the ____ the denaturation temperature
More hydrogen bonds means more intermolecular forces, which means higher denaturation temperature
higher
