exam 2 techniques

temperature sensitive mutants

an organism or gene variant whose protein product functions normally at the permissive temperature, but loses its function or changes behavior at non-permissive temperature

yeast permissive temperature

23℃

yeast non permissive temperature

36℃

complementation analysis

Designed to determine if different recessive mutations are on the same gene

restriction nucleases

discovered in 1963, found in bacteria and prevent viral infection, 800 commercially available 3000 discovered

SNPs

a difference in a single DNA building block, or nucleotide

mutations

different variant of an allele

mutation causes

UV light and chemicals

transfection

artificially introducing foreign nucleic acids into eukaryotic cells

DNA gel electrophoresis

technique used to separate and analyze DNA fragments based on their size

band/gel shift assays

technique used to detect and analyze protein-nucleic acid interactions

southern blots

gene detection - 1 copy of a gene

northern blots

gene expression, separating mRNA

molecular beacons

probes that form a hair pin like structure to detect specific nucleic acid sequences

cDNA microarrays

tool used to measure gene expression levels across thousands of genes

single cell RNA-seq

technique that measures gene expression of individual cells

genomic cloning

isolating and copying a specific segment of genomic DNA and inserting it into a self-replicating vector to create multiple identical copies

cDNA cloning

technique that converts single-stranded mRNA into cDNA and insterts it into a vector for replication and study

transfection delivery methods

microinjection, electroporation, liposomes, viral transfection

PCR/RT-PCR

amplification DNA/mRNA, heat/cooling cycles

knockout/knockin mice and cells

genome editing techniques used to study gene function and model human diseases

hESCs/stem cell therapy

uses pluripotent cells derived from the inner cell mass of blastocytes to regenerate or replace damaged tissues

siRNA

simple to make, cleaves mRNA, not permanent (transient)

shRNA

complex to make, can be permanent, more potent, cleaves mRNA

antisense RNA

less effective, bind to mRNA

CRISPR-Cas9

gene editing tool that allows scientists to alter DNA sequences and modify gene function