genetic engineering Flashcards
siRNA?
small interfering RNA.
knockDOWN - used reduce the expression of a specific gene
VNTR’s?
variable number of tandem repeats
short sequences of repeating nucleotide sequence
can be used to generate DNA profiling as vary in length in individuals
transgenic mice ?
modify mouse ES cells by southern blotting or PCR
insert modified ES into host blastocyst
into another female - chimeric mice
Knock out is mice
transgene (foreign DNA) injected into pronucleus of fertilised ova
Yeast 2 hybrid screen
to find proteins thaat interact with your protein of interest
- create 2 hybrids 1. POI and DNA binding domain 2. POI and activation domain
- if POI’s are in close proximity they will bring there binding and activating domains together and form a transcription unit
restriction fragment length polymorphism
a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases.
RNA microarray
detect expression of thousands of genes at same time
- mRNAs converted to ssDNAusing reverse transcriptase
- appplied to slide
- ssDNA will hybridise to probe cell with complementary sequence
immunohistochemistry and immunocytochemistry
histo- stains tissue sections
cyto - stains single layers of cells grown in culture
visualise location of a protein or antigen using antibodies
in situ
DNA sequencing (sanger sequencing)
primer - 15-25 bases long
aneals to DNA sequence and is extended by DNA polymerase
terminator nucleotides incorporated - lack of OH on C3
add fluorescent tag
restriction enzymes
isolated from bacteria as protect from bacteriophages
cut double stranded DNA, 4-8 bp leaving a palindromic sequence
can produce sticky ends
western blotting
detect protein after separated by electrophoresis
primary antibody binds to antigen
secondary antibody binds to primary heavy chains and has a fluorescence
can see size and relative abundance of protein
in homogenate
factors tha affect DNA migrations in electrophoresis
size - smaller DNA fragments move faster
Gel conc - DNA moves slowet in higher concentration
DNA shape- linear moves faster that circular and supercoiled faster than relaxed
gel type - agarose = 100 - 20,000bp
polyacrylamide = 10 -700 bps
ways to lyse tissue to homogenise
mechanical - pestle and mortar
chemical - detergents
repeat freeze thaw
northern blotting
visualisation and detection of RNA in homogenate
southern blotting
visualisation and detection of DNA in homogenate
chromosome painting
in situ detection of DNA - detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes
cloning uses and types of organisms used as hosts
insert DNA into host = vector using restriction enzymes
host need short life span, easy cheap to grow, vast no. , easy to break up and purity DNA from
hence use bacteria and viruses
DNA cloning process
- generate compatible cohesive ends on insert and plasmid by same enzyme
- fusion of insert using DNA ligase
- introduce recombinant DNA into bacteria
- antibiotic selction for bacteria containing
plasmid ?
small circular DNA molecule within a bacteria that is physically separated from chromosomal DNA and can replicate independently.
- contains replication origin
- has antibiotic resistance gene to select
2 types of DNA libraries
genomic - complete DNA sequence of organism
cDNA - derived from mRNA only represent expressed DNA
PCR steps and uses
- DNA amplification
- 2 to the power number of cycles
- to detect infectious agents, forensics, modifications
- template where start and end sequence known
- put in thermal cylinder with Taq polymerase and dNTPS and primers
- 95 decrees - denatures
- 55 degrees annealing of primers
- 72 degrees new strand synthesised
