GENE 314 Lab 8

single nucleotide polymorphism (SNP)

small genetic differences between individuals

causative SNP

SNPs that are correlated with a phenotype

introns

non-coding DNA in eukaryotes that’s spliced out of the primary transcript to produce mRNA

each codon encodes a(n) _______.

amino acid

wobble

third position of a codon that is flexible, allowing coding redundancy

STOP

UAA, UAG, UGA

START (Met)

AUG

restriction fragment length polymorphism (RFLP)

SNP alters the ability of a restriction enzyme to cut a piece of DNA by altering the palindrome (ex: sickle cell anemia)

what is the RFLP in sickle cell anemia

A → T in the 5th position of the palindrome

where do polymorphisms most commonly occur?

non-coding regions

what happens when polymorphisms are found in the coding sequence?

possible consequences to the encoded protein

synonymous mutation

occur in the third base codon (wobble), resulting in no change in protein expression due to the degenerate code

non-synonymous mutations (missense/nonsense)

lead to a change in the amino acid encoded in the DNA

nonsense mutation

premature stop codon resulting in a truncated protein

DNase

enzyme in the nucleus that degrades DNA and proteins that inhibit downstream analysis of DNA

InstaGene matrix

chelex matrix made of negatively charged microscopic beads that “chelate” or grab metal ions out of solution; Mg²⁺ chelated and therefore unavailable as a cofactor to DNA-degrading enzymes

why is the solution boiled before PCR?

to isolate DNA; boiling the same degrades the enzymes, but the DNA remains intact

who developed PCR?

Kary Mullis in 1983

what 5 things does PCR require?

1. DNA template

2. thermally stable DNA polymerase

3. a pair of primers

4. a mixture of the 4 DNA nucleotides

5. buffer

3 steps of PCR

denaturing, annealing, extension

what happens when DNA is heated to 94ºC (denaturing)?

hydrogen bonds between bases break, allowing the 2 DNA strands to separate

what happens when the temperature is lowered (annealing)?

primers are added to DNA

what sequence has higher annealing temperature?

high CG content (because 3 hydrogen bonds)

what occurs during step 3 of PCR (extension)?

Taq polymerase recognizes the 3’ end of the primers → temperature is increased to 72ºC → nucleotides added 5’→'3’ direction

how many strands of DNA are there after 40 cycles of PCR?

2⁴⁰ = 1.1 trillion

what is the PCR product referred to as?

amplicon

ADRB 2 (snp 2 / Gly16Arg)

associated with obesity, glucose intolerance, and elite endurance performance

PPARG (snp 4 / Pro12Ala)

transcription factor that regulate the formation of far cells; morbid obesity

ADRB2 (snp 2 (Gly16Arg) and snp 5 (Gln27Glu))

associated with asthma, obesity, and type 2 diabetes

FABP2 (srp 3 / Ala54Thr)

fatty acid binding protein

how many genes are there for bitter taste receptors in mammals?

approx. 30

what is the gene for the PTC taste receptor?

TAS2R38

halotype

one specific combination of 3 SNPs