BIMM 100: Week 4

restriction enzymes

present in bacteria, sequence-specific DNA endonucleases

complementary/sticky ends

overhanging ends that can be ligated after cleaving

Steps to Clone a Gene

-Prepare a cloning vector (self-replicating plasmid)

-Obtain DNA fragment of interest (using PCR and restriction enzymes)

-Ligate the DNA fragment and vector together to create recombinant DNA

-Transfer the recombinant DNA into a host organism (bacteria)

-Identify cells containing the desired recombinant DNA

components of replicating plasmid

replication origin (recognized by DNA helicase), selection marker, exogenous DNA cloning region

selection marker

gene on plasmid that confers specific trait such as antibiotic resistance

PCR template can be…

genomic DNA or cDNA (reverse transcribed DNA)

transformation

introducing recombinant DNA into a host cell

selection

bacteria grown on selective medium such as ampicillin plates, only cells with appropriate resistance plasmid survive

applications of recombinant DNA

gene cloning, gene inactivation, gene overexpression

recombinant protein production - A recombinant plasmid can contain…

a bacterial promoter to drive transcription/ protein-coding sequence of interest/ selection marker/ origin of replication

Cas 9

RNA-guided DNA endonuclease that introduces double stranded DNA breaks

guide RNA

contains a sequence that binds to Cas9, and a sequence that binds the target DNA

Cas9-induced dsDNA breaks can be repaired by

NHEJ (deletions) or Homologous Recombination

repetitive DNA

repeated sequences found throughout the genome

simple sequence

short repetitive DNA found in same location

mobile DNA elements / transposable elements

repetitive DNA dispersed throughout genome, can be moved to new areas by enzymes

DNA transposons

“cut and paste" as DNA, done by transposase with endonuclease and ligase activity

All transposons have…

inverted repeats (binding sites for enzymes) and a protein-coding region (product mobilizes them)

Transposition of DNA transposons during DNA replication…

may decrease/increase their copy number for a gamete

retrotransposons

Original DNA → RNA intermediate —> New DNA —> Integrated into original genome

Retrotransposons have larger … than DNA transposons

terminal inverted repeats

Transposons are kept …

mostly silent

Gene duplications give rise to…

the evolution of new protein functions

Unclassified intergenic DNA

Functionally ambiguous like repetitive DNA, potential role in gene expression and chromatin organization