DNA Replication and Sequencing Flashcards
What is the key enzyme for DNA replication?
DNA polymerase
What is the template for DNA replication?
The parent strand of DNA
What are the subunits used for DNA replication?
dNTPs
Where does DNA replication begin?
Origin of replication - ori
What type of bond links two nucleotides together?
A phosphodiester linkage between the 3’ OH of the previous nucleotide and the 5’ phosphate of the next nucleotide
What provides the energy to link two nucleotides together?
Cleavage of two of the phosphates of the dNTP
What are the 3 steps of DNA replication?
- Helicase unwinds the DNA
- Primase creates a short RNA primer
- DNA polymerase synthesizes the new strand in the 5’ to 3’ direction
What is PCR used for?
To amplify a sequence of DNA of interest in vitro
What 4 things are required for PCR?
- Template DNA of the sequence of interest
- Sequence specific primers, forward and reverse
- dNTPs
- DNA polymerase
What are the 3 steps that are repeated in PCR?
- Denaturation. The two strands of DNA are separated by heat breaking the hydrogen bonds
- Primer hybridization. Cooled to allow the primers to anneal to their complementary sequences
- Extension. The temperature is increased to the optimal temperature of the Taq DNA polymerase and it synthesizes new DNA off the primer
What can PCR tell us?
- The size of the DNA fragment between the primers
2. If the sequence of interest is present
What can’t be determined by PCR?
The sequence of DNA that was amplified between the primers
What is Sanger sequencing?
A method to determine the order of nucleotides by chain termination
How does chain termination occur with Sanger sequencing?
Dideoxynucleotides. There is no 3’ OH for DNA polymerase to extend off, so the chain gets terminated
What are the 5 components in a Sanger sequencing reaction?
- Template DNA
- dNTPs
- DNA polymerase
- Labelled primers
- One of 4 ddNTPs in small amounts
Why are the primers labeled in Sanger sequencing?
Allow them to be detected on a gel, which then allowed the elongated strand to be detected
What determines whether DNA polymerase adds a dNTP or a ddNTP?
Completely random, whatever DNA polymerase happens to grab
Why is it required to run 4 reactions for Sanger sequencing?
Each solution only has one ddNTP and will only pinpoint the positions of that particular nucleotide. Radioactivity was also used to detect the labeled primers, so we needed 4 reactions to tell the nucleotides apart
What does each band on the gel tell us in Sanger sequencing?
Read from the bottom up, it tells us which nucleotide is at a certain position past the primer
How do we do Sanger sequencing today?
We use fluorescently labelled ddNTPs and run everything out in the same solution through a capillary tube with electrophoresis. At the end of the tube, the fluorescent dye is excited by a laser and a camera detects the colour. A computer plots out the peaks of the colours, which indicates the sequence
How can PCR be applied in basic research?
Molecular cloning, genotyping, checking for mutations. Typically determining if a sequence of interest is present
How can PCR be applied in medicine?
Can be used to check for sequences of a suspected pathogen in a patient. If the pathogen is present, we will get PCR products that confirm its presence. If the pathogen isn’t there we get no PCR products
How can PCR be applied to forensics?
DNA fingerprinting. Amplifying and comparing the number of repeats in 14 short tandem repeats, which is very likely to be unique for each person
What are short tandem repeats?
A repeated sequence present at specific locations in each person’s genome, and each person has a different number of repeats
How can DNA sequencing be applied to molecular research?
Genome projects, screening during molecular cloning
How can DNA sequencing be applied to organismal research?
Biome sampling, fossil identification, building phylogenies
What are some benefits of personal genome sequencing?
Personalized medicine, knowledge of disease predisposition, it’s cool
What are some downsides of personal genome sequencing?
Do you actually want to know if you’re predisposed to a disease, genetic privacy
