PCR and individual variation Flashcards
What is PCR?
A method used to detect specific genetic sequences in the presence of billions of bases of human DNA. It does this by rapidly copying the desired segment of DNA.
What does PCR require?
1) DNA template - segment of DNA to be replicated
2) Primers - short pieces of DNA complimentary to part of the DNA template
3) Free deoxyribonucleotide triphosphates (DNA nucleotides) - to add to the growing daughter strand
4) Enzyme - to catalyse the process (Taq polymerase)
5) PCR buffer - to maintain an appropriate pH for the enzymatic activity
What are the three steps of PCR?
Denaturation, annealing and extension
What does denaturation involve?
DNA strands are denatured (separated) using heat 95*C
What does annealing involve?
Primers (small complimentary segments of DNA) anneal to the separate strands at specific locations determined by hydrogen bonding between complimentary nucleotides.
How many primers do you need when replicating a segment of DNA?
2 - one for each strand
What does extension involve?
The enzyme Taq polymerase extends the primers, facilitating synthesis of new DNA from complimentary free nucleotides (order determined by the template DNA) and resulting in twice as many strands of the original DNA.
Why is the PCR process repeated?
It is repeated in a thermal cycler every few minutes to create millions of exact copies very quickly. After about 30 cycles, there are over 1 billion copies of the target sequence.
What was the old process of PCR?
Used the enzyme DNA polymerase so step 3 had to be performed at 37C.
This enzyme denatures at high temperatures (90C) so each cycle necessitated the addition of more enzyme –> inefficient and expensive
What is the new process of PCR?
Uses a different enzyme Taq polymerase from thermophilic bacteria. Stable at 98C, works at 70C.
This means that no enzyme has to be added between cycles (cheaper and more efficient) there is also less need for heating and cooling.
What is the use of electrophoresis?
DNA synthesised through PCR can be analysed by separating the DNA fragments by DNA gel electrophoresis.
How does electrophoresis work?
The gel is made up of a web-like matrix that is set. As the DNA moves through this matrix, shorter strands are able to move more easily than longer strands - they separate.
DNA is made to move because an electric current is applied to the gel (positive electrode on the other end to the DNA). DNA has a negative net charge, so is attracted towards the positive pole. A high voltage (100V) is used.
The DNA, mixed with a fluorescent dye, is added to preformed wells at the end of the gel.
Once the DNA has separated, the gel plate can be placed under UV light (in a gel dock), where the fluorescent dye begins to glow. This allows the DNA bands to be visualised and captured by a computer.
What are some uses of PCR?
Disaster identification, forensic testing, paternity testing.
What are SSRs?
Simple Sequence Repeats - areas of DNA that are constituted by repeating 3 nucleotide sequences.
How can PCR be used as genetic testing for specific diseases?
By using primers that anneal to areas
immediately adjacent to specific genes, scientists are able to determine whether a person has an intact copy of a gene or not - can provide information about disease.
