Chapt 10 Flashcards
genetic engineering
- using recom DNA for :
- manipulating genes for analysis
- develop products
Gene therapy
- replace/repair gene
- use ‘altered’ viruses to insert into host
- performed on somatic cells
- germ line therapy
- targeted gene therapy
Gene Therapy considerations:
- gene must be ID and cloned
- what mutation is causing disease
- must insert “good” copy somewhere in euchromatin genome ***only masks non-functioning gene
Germ line vs Somatic therapy
- benefit offspring only
vs
-only humans and benefits individual ONLY
Targeted gene therapy
- swap out abnormal w/ normal gene
- homologous recom
- SMALL % of success
Biotechnology
-produce useful commercial products via genetic manipulation
Bioluminescence
natural vs synthetic
- specialized vector
- screening with probes
Specialized vectors
- type
- features
-shuttle : introduced into multiple organisms
-expression: derivative of plasmid vector and is allowed to transcript/translate into cloned genes
1Promoter:upstream of MCS
2Transcription terminator: downstream MCS
3*Shine-Delgarno sequence: translation initiation–> ribosomal binding site in mRNA
Issues when making expression clones
- getting insert in correct orientation (50/50 chance) :
1. ) sequence DNA and check
2. )Restriction mapping
3. ) directional/forced cloning (PCR designed primer)
Restriction mapping steps
- digest DNA w/ various Restriction enzymes
- electrophrese digest on agarose
- measure band frag sizes
- determine ORDER of band
Probes:
- requires?
- types
- ssDNA
- lablled and tagged : radioactively OR non-radi(fluorescent)
- Heterologous: dna sequence from dif BUT related org
- Oligonucleotide: DNA articially synthesised
- cDNA: based on mRNA sequence
Probe steps
- Transform DNA
- plate on selected media
- transfer transformED colonies (white) to same media
- velvet replicat plate
- filter paper on rep plate
- remove filter paper, lyse cells and bind ssDNA to filter paper
- f.p. in zipblock w/ DNA Probe
- Incubate/hybridization
- wash fp to remove excess probe
- ID probe: GOI
- locate match from origainal plate
DNA polymorphisms
- def
- types
- useful as DNA marker: 2+alleles @ 1 loci
- # repeated NT units
- Single NT polym (SNP):bp dif b/n ind==> abolish or create restricition sites
- Short Tandem Repeat( STR): microsatelites (very short, ind can be heter or homo for STR)
- variable # tandem repeats(VNTR): minisatelites
Variable number tandem repeats (VNTR) process
- DNA digest w/ restrictions enzyme thats flanking VNTR
- fragments are electrophoresed +blotted to filter (Southern Blotting)
- Blot probed w/ VNTR repeating sequence
Concept of DNA molec testing
- designing test require knowledge of gene mutations
- reveal presence of mutat assocated w/ genetic disease
