DNA replication Flashcards
(15 cards)
What is DNA replication and what is it used for?
It is the production of an exact copy of a DNA molecule. With identical base sequences
Used for:
reproduction: passing hereditary information to offspring.
Growth and repair - growth and tissue replacement: process of cell division to create new cells (requires repliaction of DNA)
How is DNA built? (1/2)
It is built by adding and linking nucleotides together.
Nucleotides are complementary - and only joined together in one way:
Adenine to Thymine
Guanine and Cytosine
nr of hydrogen bonds formed is different.
G to C is 3
A to T is 2
Complementary base pairing leads to high accuracy in replication.
How is DNA replication carried out?
By an assembage of subunits called replisome.
Enzyme helicase breaks the hydrogen bonds between the two strands of DNA.
The helicase will unwind and unzip the DNA, thus reparating the strands into single strands.
How is DNA copying carried out (2/2)
At the replication fork, which is created by the helicase.
DNA polymerase will move along the single strands, adding and fusing nucleotides into new strands complementary to the original (called template strands).
1 hydrogen bond is created to the template strand. Then a covalent bond between the nucleotide and the end of a new strand. The phosphate in free nucleotide connects with sugar in the strand.
Semi-conservative DNA
The DNA is considered semi-conservative because each DNA molecule consists of one original (parental) strand and a newly synthesized strand
Since the bases are complementary the two double stranded DNA- molecules are identical copies.
What is PCR?
Polymerase chain reaction.
An automated method of DNA replication. It allows for the copying of the gene or part of DNA of our choice - it amplifies the DNA. Meaning increasing the size of the sample.
How does PCR work?
It uses primers. Primers are short segments of DNA, to signal when to start copying. By selecting a proper primer, it allows us to select the section of DNA to be copied.
Primers are short sequences that DNA-polymerase attaches to, to initiate copying of the DNA.
The process of PCR?
- Denaturating, the temperature is set to 95 degrees, which breaks the hydrogen bonds and separating the strands.
- Annealing - cooling to 45 degrees allows primers to bind.
- Elongation - Heating to 72 again, which is the optimum for Taq DNA polymerase to build new strands.
- Process repeated 30 times. Each cycle, the quantity of DNA is doubled.
what is Taq DNA polymerase?
a bacteria, which is used because heat would normally denature enzymes. Taq is adapted to warm conditions and heat tolerant.
Helicase
an enzyme that breaks the hydrogen bonds between the bases to separate the parent strands unwind the double helix.
DNA polymerase
the enyme that adds free nucleotides to the new strand, creating a bond between the phosphate of the free nucleotide and the sugar of the last nucleotide on the strand.
Gel electrophoresis?
A method to separate DNA fragments by lenght.
How does gel electrophoresis work?
A gel with wells is submerged in a buffer in a tank with electrodes on each side.
When an electric field is applied, DNA starts to migrate towards the positive pole.
Small pieces migrate faster than large pieces due to less collisions with gel
Paternity testing
When testing a person, non-coding sections of DNA is used - contains information about health.
These sections contain short repeating sections, STR’s. There is different number of repeats in this sections between persons.
DNA from STR’s are amplified with PCR and sorted with gel electrophoresis.
Testing for coronavirus
- get the virus particles, which RNA will be tested.
- Convert RNA to DNA using enzyme reverse transcriptase.
- Amplify RNA with PCR
- Add fluroscent marker that can attach to DNA
- If level of fluroscence rises above threshold the person is positive.
