MCB 250 exam 3

Northern Blot

Extracted RNA from tissue, then denatures RNA, gel electrophoresis, transfer to membrane, throw in probe, wash off probe, specifically for RNA and is used to measure gene expression through mRNA levels. DOES NOT NEED PRIMER

Southern Blot

Extracted DNA from tissue, then denatures DNA, gel electrophoresis, transfer to membrane, throw in probe, wash off probe. Specifically for DNA, used to detect specific gene sequences and gene mapping. DOES NOT NEED PRIMER

Western blot

specifically for proteins. Uses polyacrylamide gel and an electric current to transfer to membrane. Uses specific primary or secondary antibodies instead of a probe. Used for identifying specific proteins or confirming diagnostics. DOES NOT NEED A PRIMER.

Sanger Sequencing

Requires a primer and a fluorescently labelled nucleotide. DNA polymerase extends the strand using thrown in dNTPs until a ddNTPs is added which terminates elongation. Fragments are sorted through gel electrophoresis and identified by the fluorescently labelled nucleotides.

Next generation Sequencing

DNA molecules are prepared for sequencing in cell-free reactions
Millions of individual DNA fragments are isolated and sequenced in parallel during each machine run
Advanced fluid-handling technologies, cameras, and software make it possible to detect sequencing products in extremely small reaction volumes.

How do you read a gel when looking for the sequence of the newly synthesized DNA

From bottom to top, and it is the exact nucleotides that are displayed

How do you read a gel for the template strand

From top to bottom, and it’s the complementary base pair.

Homologous Recombination

Found primarily in Eukaryotes. Essentially a DNA rearrangement that includes Holliday Junctions. DNA synthesis is necessary for this. Primarily found when the replication fork collapses. Can lead to Crossover in Meiosis.

Enzymes necessary for Homologous Recombination

RecBCD, RecA, RuVA, RuVB, and RuVC

RecBCD

Unwinds and degrades the DNA until it hits a Chi site which promotes it to leave a 3’ overhang. Chi site has directionality

RecA

Finds single-stranded DNA and binds to it, coating the entire strand, then finds the homologous sequence on another chromosome sand causes strand invasion. Sets up the Holliday junction

RuvAB

Two proteins that work together to migrate the holiday junction across the strands. RuVB has helicase activity that uses ATP to move the DNA strand through RuvA.

RuVC

An enzyme that cuts the holliday junction in half, producing either a crossover or patch.

Non-Homologous End Joining (NHEJ)

A DNA repair system primarily used in higher level eukaryotes and is not found in e.coli. Essentially uses proteins that “clean up” the DNA after it has been cleaved. Sometimes it cleans too much and ends up degrading important information from the DNA, causing mutations in the gene.

Translocation

Basically, in chromosomes when some portion of the DNA is broken off and then swapped. This can happen between any two chromosomes

CRISPR and NHEJ

CRISPR is essentially when we introduce RNA guides to DNA to create double-stranded breaks. This happens because the RNA guides Cas9 to the specific site and then has the endonuclease cleave the area. A gene can be knocked out when NHEJ tries to repair the ends and ends up deleting important information from the gene, making it lose its function.

CRISPR and Homologous Recombination

After Cas9 cleaves the region, homologous recombination can be used to synthesize the rest of a region that was previously missing using a template, essentially fixing a gene.