BIO 1511 - PCR & Gel Electrophoresis

What is PCR and what does it mean?

Polymerase chain reaction; detects specific sequence of DNA, amplifies section of DNA targeted, and creates millions of copies of it by using repeated cycles of heating and cooling 

What are alleles?

Copies of genetic material that contain code for characteristics; some don’t code for anything but are within DNA sequence and can be extracted

How are humans different?

Genetic composition is the same, but variable expression of genetically controlled traits 

Characteristics of Alu Allele and what it is/where

Alu elements are located in PV92 region of chromosome 16; alu alle is a repetitive element in the human genome within introns (no code for protein), and are ~300 base pairs long. 

Can be heritable and dimorphic 

Whats dimorphic?

some families display alu elements on chromosome 16 and some don’t

some have and some don’t

What are the Alu genotypes? 

Individuals can be homozygous positive or negative (both have PV92 Alu or not) OR individuals can be heterozygous

How does presence of alu genotype get detected?

Can be determined by size of extracted DNA (# of base pairs)

What is the first step of process of PCR? and steps?

To provide sample test for target DNA sequence it is DNA EXTRACTION

1. Collect cheek cells via swishing with saline solution then incubated at 56C to soften cell membranes

2. Incubate cells to release DNA from cell and nucleus (incubated at 100C and centrifuged to release DNA from nuclei)

3. Separate DNA template (DNA want to amplify) from supernatant (fluid containing other contents of cell etc)

What are the 3 parts of the PCR?

1. Denature sample DNA (sample heated to 94C to separate two strands of DNA)

2. Anneal primers to new strand (sample cooled to 60C/37C for primers to attach)

3. Extend new nucleotides to complete new strand of DNA (sample heated again to 72C and heat tolerant enzyme, DNA polymerase adds free nucleotides to extend new, complementary strand of DNA)

Repeat for 40 cycles to amplify strand

What polymerase is used extensively for PCR? 

Taq polymerase originally identified in hot springs in Yellowstone National Park, adapts to high heat environments and is stable through multiple cycles of heating— 

What happens at end of PCR cycle process?

Two copies of original DNA fragment are produced — several more cycles follow doubling # of fragments each time

Equation for PCR Amplification

2^n where n= # of cycles

2^40 = 1.1×10^12 (over 1 billion+ copies)

What is added during DNA extraction in the experiment and what does it do/contain?

InstaGene Matrix added to expressed saline solution — contains - charged microscopic beads that bind to + charged ions such as Mg+ in solution.

Without matrix, cellular enzymes that require Mg2+ would degrade the DNA after it’s released from nucleus. After incubation the beads are pulled from solution through centrifugation and the supernatant (liquid solution) that contains DNA template without risk of degradation by enzymes

Why is it important to avoid transfer of matrix into PCR tube? 

Because the matrix contains many substances that block the DNA polymerase; can be used to bind/deactivate Taq polymerase, stopping amplification INHIBITS PROCESS 

If it enters PCR tube, it will remove Mg+ PCR mix and stop Taq Poly

What is contained in Master Mix and components are needed to?

• Free nucleotides, primers, and DNA polymerases; contained in Master Mix added prior to PCR

• During heating/cooling cycles in thermal cycler, all components needed to denature DNA, anneal primers, and extend strand in PCR tube.