E7 Quiz

What makes it work?

+ DNA thermal cycler + Taq DNA polymerase! + Taq was isolated from Thermus aquaticus, a bacteria that grows in hot springs (~75oC) - This organism's enzymes have adapted to the high temperature, so they can survive cycling through the high temperatures;

What factors influence the rate of migration in DNA agarose gel?

+ Molecular size of the DNA The mobility of linear DNA fragments is inversely proportional to the log10 of their molecular weight. + The conformation of DNA + The agarose concentration + The buffer + The applied voltage

Mitochondrial DNA Template

+ The mtDNA Genome + Comparing mtDNA with nuclear DNA + The Expected PCR Product - Where are the 2 primers? - What is the expected size of the PCR product? - Sequence variations: single nucleotide polymorphism (SNPs)

The purpose of PCR

+ Within a few hours, greatly amplify unique regions of DNA so they can be detected in large genome + This amplified sample then allows for size determination, restriction mapping and nucleotide sequencing Basic Research Applied Research + DNA "fingerprinting"--forensic science + Paternity determination + Disease diagnosis

What two components need to be added to the puReTaq Ready-To-Go PCR Beads to set up a PCR reaction?

1) a new pair of primer/ loading buffer mixed 2) DNA template (from yourself)

Primers

1. A pair: 2 primers 2. Usually about 20 nucleotides in length 3. Designed to flank the region to be amplified 4. Must be complementary to the 3' end of the gene to be amplified 5. Quantity: In large excess

Extracting and Amplifying mtDNA

1. Polymerase chain reaction (PCR) 2. Mitochondrial DNA 3. Experiment 7 content

The basic protocol

1. Step 1: denaturation of DNA 2. Annealing of primers to DNA 3. Extension by polymerase 4. Repeat 30-35 times

The reaction mixture

1. Target DNA (purified or a crude extract) 2. A pair of primers specific for the target DNA 3. Free deoxynucleotides (dATP, dGTP, dCTP, dTTP): building blocks/energy source 4. Heat stable DNA polymerase (Thermus aquaticus) 5. Buffer (containing cofactor, Mg++)

What determines the size of the final PCR product?

2 locations of the 2 primers to find the size

What temperature is used to synthesize the new DNA strand?

72C - 75C

What temperature is used to denature the DNA template?

95C

Single Nucleotide Polymorphism (SNP)

A small genetic change, or variation, that can within a person's DNA sequence + AAGGTTA + ATGGTTA SNPs are stably inherited in a Mendelian fashion-used to study human diversity and evolution

primer design PCR can anneal to DNA of both human epithelial cells and bacteria collected in mouthwash

False, it only recognize human cell

What is unique about the Taq DNA polymerase?

Heat resistant, thermostability

What would be the consequence if the reaction contained DNases (enzymes that can degrade DNA into nucleotides)?

If DNases were present, those enzymes would degrade the DNA components, then PCR would fail also

What would be the consequence if the reaction contained proteases (enzymes that can degrade proteins into amino acids)?

If proteases were present, those enzymes would degrade Taq, thus PCR would fail

Do you need to have a pure template DNA sample (free of other DNA sequences) to allow specific amplification of a segment of the template DNA?

No because PCR only works for human DNA (they have been programmed specifically)

E7 content

Part 1: total DNA isolation Part 2: mtDNA amplification by PCR Part 3: PCR product analysis by gel electrophoresis

5' CATGGATG 3' is complementary to 5' GCATGAATGCATGCA ... TGCATCCATG3' so it can be used as one of the primers in pCR to amplify this DNA region

True

The amplification of the original target DNA is exponential.

True

The PCR machine

Very rapidly changes of the temperature between the various stages of the PCR process Programmable for use with many different cycling parameters

What is the direction of synthesis of the new DNA strand in each cycle?

from 5' to 3'

Primers need to be designed ...

outside