Lab 8: Single Nucleotide Polymorphisms

What is a SNP?

single nucleotide polymorphism

• small variations, often one base pair, that exist between individuals

SNPs that are correlated with a phenotype are called

causative SNPs

What are introns?

the primary transcript from the DNA that contains extra pieces of information

What is the fate of an intron?

spliced out of the primary transcript to produce a mature message, or mRNA, that is then translated

What is the start codon in most organisms?

AUG - codes for methionine

The 3’ end is also called the:

third or wobble position

How does a SNP affect a restriction enzyme?

alters a restriction enzymes ability to cut a piece of DNA by altering the palindrome

• this is the case in the gene responsible for sickle cell anemia

What is a SNP called when it alters a restriction enzymes ability to cut DNA?

Restriction Fragment Length Polymorphism (RFLP)

What is the difference between the DNA sequence of a non-sickle cell individual vs a sickle cell individual?

non-sickle cell individual: CCTGAGG

sickle cell individual: CCTGTGG

Where do polymorphisms occur?

most often occurs in non-coding regions but can occur in coding regions

• effects of SNPs will vary depending on where they occur

Within a coding region, what is the effect of a SNP

might lead to the same or an equivalent amino acid substitution

What are the effects of a SNP outside of the coding region?

there are many regulatory regions that if altered might change the expression level of one or more proteins

Which mutation occurs in the third base of a codon, or the wobble position?

Synonymous mutations

What is the result of a synonymous mutation?

no change to the protein because the same amino acid is encoded

• thats why it’s called the silent mutation

What’s an example of a synonymous mutation?

changing CAA to CAG

Which mutation leads to a change in the amino acid encoded in the DNA?

non-synonymous mutations

What is the result of a non-synonymous mutation?

a change to the structure of the protein and by extension alter the function

What’s an example of a non-synonymous mutation?

changing CAA to CGA

What is a nonsense mutation?

another type of a non-synonymous mutation that occurs when the altered codon does not code for any amino acid

• in other words, it’s a stop codon

What is the result of a nonsense mutation?

a truncated or shortened protein which can have drastic affects on the protein’s function

What is an example of a nonsense mutation?

changing CAA to UAA

Identify the stop codons.

UAA, UAG, UGA

What is an important aspect of DNA extraction?

separating DNA from the cellular “junk” (enzymes such as DNase, other proteins, and cellular debris)

What is the InstaGene matrix?

a chelex matrix made of negatively charged microscopic beads that “chelate” or grab metal ions out of a solution

Which metal ion does the InstaGene matrix chelate specifically?

Mg2+ → required as a cofactor in enzymatic reactions

Why is Mg2+ specifically important to grab?

makes it unavailable to the enzymes that are also present and would degrade the DNA you are trying to extract

What happens when you boil the samples?

it degrades the enzymes, but the DNA will remain intact and ready for Polymerase chain reaction (PCR)

Who developed PCR? What year?

Kary Mullis; 1983

What are the five things required in the PCR process?

• DNA template - generated from DNA extraction in the first step

• DNA polymerase (thermally stable)

• A pair of primers

• mixture of the four DNA nucleotides (ATGC)

• Buffer for stability and activity of the enzyme

Why is a thermally stable DNA polymerase required in the PCR process?

because the PCR reaction is heated to 94 degrees C

• destroys the biological activity of most enzymes

Why is a pair of primers required in the PCR process?

they will recognize specific sequences in a template consisting of billions of base pairs

• primers (aka oligonucleotides) are short strands of DNA (18-30 bases in length)

• designed to be complementary to a region of interest and are strand specific

Why is a mixtures of DNA nucleotides required in the PCR process?

as the primers anneal to the template, the polymerase extends the primers by incorporation of the nucleotides

PCR consists of what three steps?

denaturing, annealing, and extension

Purines are paired with pyrimidines with what type of bonds?

hydrogen bonds

Describe the denaturing step in PCR

Solution is heated to 94 degrees C to break the hydrogen bonds between the purines and pyrimidines

Describe the annealing step in PCR

the temperature is lowered to allow annealing of the primers.

• temperature is specific for each set of primers and is mathematically calculated based on the sequence

• longer primers or primers with a higher GC content will generally have higher annealing temperatures

Describe the extension step in PCR

Taq polymerase recognizes the 3’ end of the primers in the duplex that has formed.

• the temperature is increased to 72 degrees C which is the optimal temperature for polymerase activity

• nucleotides are added in a 5’ to 3’ direction

Taq DNA polymerase is isolated from what?

thermophilic bacterium, Thermus aquaticus

• found in high temperature steam vents such as those found in Yellowstone National Park

How many copies of DNA would you expect at the end of 40 cycles?

1.1 trillion copies (2⁴⁰)

The PCR product can be analyzed through:

restriction digest and/ or gel electrophoresis

Out of billions of base pairs of human DNA, approximately _% varies between individuals

0.1%

• this is why people react differently to drugs