BIOL408, Unit 1: genetic engineering and omic studies

what do most methods of genetic engineering target?

protein level

vector

a virus or plasmid that is self-replicating

what do vectors contain?

and origin of replication, cloning site, promotor region, and selection marker

selection marker

tells you where the target gene is present in specific cells

cloning site

place where gene of interest is inserted, in place of a restriction enzyme

what are the 4 main methods of genetic engineering?

PCR, homologous recombination, RNAi, and CRISPR

PCR

mass replication of sequences for analysis

what is needed for PCR testing?

DNA sequence, primers, DNA pol, free nucleotides, and a buffer

what are the steps for PCR testing?

denaturation, annealing, and extension

homologous recombination

genes/sequences are inserted into a cell

RNAi

interference of transcription to reduce gene expression

how is RISC involved in RNAi?

it temporarily binds to target mRNA sequences which leads to cleavage, disrupting transcription

what is the outcome of RNAi?

gene knockdown, or temporary silencing of gene expression

what are the types of omic studies?

genomic, transcriptomic, proteomic, and metabolomic

genomic studies

examine DNA sequences, two kinds: sanger and next generation

sanger sequencing

hijacks DNA to read short sequences

what are the characteristics of sanger sequencing?

good for small sequences or single genes, can only read one strand at a time

new generation sequencing

uses a reference sequence to generate read sequences

what are the characteristics of new generation sequencing?

can sequences 1000s of genes at once, faster and less expensive for large sequences, can read an entire genome

transcriptomics

examination of RNA sequences expressed or present in a cell

what are the types of transcriptomics?

microarray and RNA seq

what are the forms of transcriptomics?

microarray and RNA seq

microarray

identifies transcripts expressed in different cell or tissue types

how does microarray work?

uses fluorescent label probes to target known sequences

RNA seq

uses next gen sequencing

proteomics

examines proteins expressed in a cell or tissue at any given time

what are the types of proteomics?

immunoassays, western blot, and mass spectrometry

immunoassays

uses antibodies to label proteins and reveal their location, but not quantity

what are the two kinds of immunoassays?

immunocytochemistry and immunohistochemistry

western blot

separates proteins based on molecular weight, interactions, or quantity

mass spectrometry

separates proteins by size, charge, structure, or interaction by ionizing protein fragments