Transcript PCR Lab Notes

PCR Lab Notes
What does PCR Stand For?
Polymerase chain reaction
Who invented PCR?
Kary Mullins
Four main areas of biotechnology




Gene mapping
Cloning
DNA sequencing
Gene Detection
What is the function of PCR?

PCR produces exponentially large
amounts of a specific piece of DNA
from trace amounts of starting
material.
The steps of PCR
1.
2.
3.
Denature the double strand using
heat (94 degree Celsius).
Anneal Primers – Cool temperature
down to 60 degree Celsius.
Polymerization – Increase
temperature to 74 degree Celsius.
Genes and DNA
What you already know….




There are 23 pairs of chromosomes which
contains 30,000 to 50,000 genes.
These genes only comprise about 5 % of
chromosomal DNA.
The other 95% is non-coding DNA.
The sequence with the genes are introns,
which is transcribed into RNA but in the
end do not make a protein.
What you already know cont…


The sequence that do code for
proteins are called exons.
Both introns and exons are initially
transcribed, then introns are spliced
out of the RNA to create the
messenger RNA (mRNA).
About the exons and introns



Exon sequences are similar among
individuals.
Introns vary in size and number
among individuals.
It is the difference in intron
sequences that allows us to
determine human genetic diversity.
The Alu Sequence


The Alu sequence is about 300 base
pairs long that is repeated, one copy
at a time, almost 500,000 times
within the human genome.
The origin and function of the Alu
sequence is not yet known.
The Alu sequence cont…

Individuals can be:
Genotype
Size of PCR
Products
Homozygous
(+/+)
Homozygous (-/-)
941 base pairs
Heterozygous (+/-)
941 base pairs and
641 base pairs
641 base pairs
The Hardy-Weinberg
Theorem
The Hardy-Weinberg equation
p2 + 2 pq + q2 = 1
Where:
p and q represents alleles.
 p2 = the expected frequency of the homozygous
(+/+) genotype in the population.
 2pq = the expected frequency of the
heterozygous (+/-) genotype in the population.
2
 q = the expected frequency of the homozygous
(-/-) genotype in the population.
The Hardy-Weinberg equation

This equation describes the
frequencies of genotypes in a
population that is at “genetic
equilibrium,” meaning that the
frequencies are stable from
generation to generation.
The Hardy-Weinberg Theory

The theory states that for a
population to achieve this
equilibrium, the population must be:
• Very large
• The members must mate randomly and
produce offspring with equal success
• there must be no migration of
individuals into or out of the population.