PCR Lab NotesPCR Lab Notes

What does PCR Stand For?What does PCR Stand For?

Polymerase chain reactionPolymerase chain reaction

Who invented PCR?Who invented PCR?

Kary MullinsKary Mullins

Four main areas of biotechnologyFour main areas of biotechnology

Gene mapping Gene mapping CloningCloning DNA sequencingDNA sequencing Gene DetectionGene Detection

What is the function of PCR?What is the function of PCR?

PCR produces exponentially large PCR produces exponentially large amounts of a specific piece of DNA amounts of a specific piece of DNA from trace amounts of starting from trace amounts of starting material.material.

The steps of PCRThe steps of PCR

1.1. Denature the double strand using Denature the double strand using heat (94 degree Celsius).heat (94 degree Celsius).

2.2. Anneal Primers – Cool temperature Anneal Primers – Cool temperature down to 60 degree Celsius.down to 60 degree Celsius.

3.3. Polymerization – Increase Polymerization – Increase temperature to 74 degree Celsius. temperature to 74 degree Celsius.

Genes and DNAGenes and DNA

What you already know….What you already know….

There are 23 pairs of chromosomes which There are 23 pairs of chromosomes which contains 30,000 to 50,000 genes.contains 30,000 to 50,000 genes.

These genes only comprise about 5 % of These genes only comprise about 5 % of chromosomal DNA.chromosomal DNA.

The other 95% is non-coding DNA.The other 95% is non-coding DNA. The sequence with the genes are introns, The sequence with the genes are introns,

which is transcribed into RNA but in the which is transcribed into RNA but in the end do not make a protein. end do not make a protein.

What you already know cont…What you already know cont…

The sequence that do code for The sequence that do code for proteins are called exons.proteins are called exons.

Both introns and exons are initially Both introns and exons are initially transcribed, then introns are spliced transcribed, then introns are spliced out of the RNA to create the out of the RNA to create the messenger RNA (mRNA). messenger RNA (mRNA).

About the exons and intronsAbout the exons and introns

Exon sequences are similar among Exon sequences are similar among individuals.individuals.

Introns vary in size and number Introns vary in size and number among individuals.among individuals.

It is the difference in intron It is the difference in intron sequences that allows us to sequences that allows us to determine human genetic diversity.determine human genetic diversity.

The Alu SequenceThe Alu Sequence

The Alu sequence is about 300 base The Alu sequence is about 300 base pairs long that is repeated, one copy pairs long that is repeated, one copy at a time, almost 500,000 times at a time, almost 500,000 times within the human genome. within the human genome.

The origin and function of the Alu The origin and function of the Alu sequence is not yet known. sequence is not yet known.

The Alu sequence cont…The Alu sequence cont…

Individuals can be: Individuals can be:

GenotypeGenotype Size of PCR Size of PCR ProductsProducts

Homozygous Homozygous (+/+)(+/+)

941 base pairs941 base pairs

Homozygous (-/-)Homozygous (-/-) 641 base pairs641 base pairs

Heterozygous (+/-)Heterozygous (+/-) 941 base pairs and 941 base pairs and 641 base pairs641 base pairs

The Hardy-Weinberg The Hardy-Weinberg TheoremTheorem

The Hardy-Weinberg equationThe Hardy-Weinberg equation

pp2 2 + 2 pq + q+ 2 pq + q2 2 = 1= 1Where:Where:p and q represents alleles.p and q represents alleles. pp2 2 = the expected frequency of the homozygous = the expected frequency of the homozygous

(+/+) genotype in the population.(+/+) genotype in the population. 2pq = the expected frequency of the 2pq = the expected frequency of the

heterozygous (+/-) genotype in the population.heterozygous (+/-) genotype in the population. qq22 = the expected frequency of the homozygous = the expected frequency of the homozygous

(-/-) genotype in the population. (-/-) genotype in the population.

The Hardy-Weinberg equationThe Hardy-Weinberg equation

This equation describes the This equation describes the frequencies of genotypes in a frequencies of genotypes in a population that is at “genetic population that is at “genetic equilibrium,” meaning that the equilibrium,” meaning that the frequencies are stable from frequencies are stable from generation to generation. generation to generation.

The Hardy-Weinberg TheoryThe Hardy-Weinberg Theory

The theory states that for a The theory states that for a population to achieve this population to achieve this equilibrium, the population must be:equilibrium, the population must be:• Very largeVery large• The members must mate randomly and The members must mate randomly and

produce offspring with equal successproduce offspring with equal success• there must be no migration of there must be no migration of

individuals into or out of the population. individuals into or out of the population.