Genomic Technologies Flashcards
What are restriction endonucleases?
enzymes that cleave DNA at specific sequences
E.g. EcoRI
What is gel electrophoresis?
A way of visualizing DNA that separates fragments based on size
What is recombinant DNA?
Any form of linking two initially separate pieces of DNA.
E.g. adding sequences to vectors
What is molecular cloning?
A DNA fragment is inserted into a vector that can replicate independently in a host cell
What are three kinds of vectors designed to accommodate large DNA inserts?
Cosmids
Bacterial Artificial Chromosomes
Yeast Artificial Chromosomes
How is RNA cloned?
By using reverse transcriptase to copy it into DNA. The resulting DNA (cDNA) is ligated to a vector DNA
What is Whole Exome Sequencing?
DNA sequencing of all of the coding exons in the genome
What is RNA sequencing?
Characterizing the transcriptome by sequencing mRNAs.
What is Chip Sequencing?
Sequencing the binding sites for different transcription factors (DNA protein interaction)
What is Genomic medicine?
Using a patients DNA sequence for monitoring and guiding therapy
Tumor therapy
How can sequencing be utilized with epigenetics?
Assessing the epigenome using bisulfite sequencing.
Allows detection of fetal syndromes
What is metagenomics?
The assessment of the microbiome using DNA sequencing
What is the significance of the 16s rRNA gene in metagenomics?
The 16s rRNA is highly conserved, but has variable regions that are species specific. Allows study of evolutionary relationships and ID
What is nucleic acid hybridization?
DNA strands are initially separated by high temperature, and then re-form helices when cooled
What is Southern blotting?
Cloned DNA is labeled with radioactive NTs or fluorophores.
Labeled DNA is then used as a probe that hybridizes with complement DNA or RNA
What are DNA Arrays?
DNA “microchips” used to detect gene expression levels or SNP detection
What is PCR?
An in vitro method for the enzymatic synthesis of specific DNA sequences
What three steps are repeated in PCR?
Denaturation
Annealing
Extension
What is Real-time PCR or Quantitative PCR?
The formation of PCR product is monitored continuously by means of fluorescent primers
Utilized in mRNA analysis for gene expression
What are four applications for PCR?
- DNA amplification
- Assessing Gene expression
- Genotyping
- in situ PCR
What is in situ PCR?
The amplification of the target nucleic acid sequences in cells or tissue samples that are fixed and permeabilized.
Assesses if and where a gene is expressed
How can gene function be studied in eukaryotes?
By introducing cloned genes into the germ line of multicellular organisms.
E.g. Transgenic mice
What is gene knockout?
Eliminating the activity of the normal gene copy to determine its role.
What is in vitro mutagenesis?
Inactivating certain genes in mouse ESCs, which grow into mice where the effect of the inactivation can be studied.
What is the Cre-Lox system?
Takes advantage of a site=specific recombinase to introduce gene deletions, inversions or translocations.
Allows for conditional mutagenesis
What is the CRISPR/Cas
Allows for targeted gene editing.
What are MicroRNA?
Highly conserved mechanism that targets mRNAs in the 3’ UTR to regulated protein expression post-transcriptionally
What are small interfering RNAs and how do they work?
Inhibits mRNA by activating endonuclease that digests a dsRNA target containing mRNA
