MCBL Lab Lecture 4

16s Ribosomal RNA

• Can be aligned across species

• Vertically transferred

• Universal molecule - translation

• Conservered function - translation.

Constant substitution rate (16s RNA)

Sequence divergence porportional to time.

Conserved regions (16s RNA)

Allows correct alignmnet and primer design

Variable regions (16s RNA)

Used as a molecular identifier.

PCR & 16s gene

Amplify to sequence and compared to databases to identify microorganisms.

PCR Melting

Break hydrogen bonds keeping DNA double stranded, allowing for each strand to act as a template.

PCR annealing

Binding of primers to template strands

PCR extension

DNA polymerase extends DNA from primers based on the sequence of the template.

PCR (After amplification)

PCR DNA product is cleaned to remove primers w/ ExoSAP

Concentration check

Quality-check for protein & salt contamination.

Spectrophotometer measurement of concentration & contamination

Quantify rRNA genes by measuring absorbance at 230-280 nm. Contaminants like proteins absorb at those wavelengths, thus giving us a measure DNA purity.

What does ExoSap not remove in dna?

Proteins

What is Sanger Sequencing?

Method of sequencing DNA using di-deoxy nucleotide terminators.

Normal dNTPs retain the 3’ -OH for extension

Incorportaion of a ddNTP will end an extension reaction

What if you have too much ddNTP in sanger sequencing?

Not enough bands in sequencing.