DNA recombination and technology Flashcards
Restriction enzymes
endonucleases produced by bacteria
recognize specific 4-to 8-bp short
palindromic sequences(restriction sites)cleave both DNA strands at this site.
It recognizes and cleave foregin DNA
protects DNA from the DNA of foreign organisms by digestion
PCR-RFLP
Restriction Fragment Length Polymorphism
inherited difference in homologous DNA detected with fragments of different length
DNA library
Fragments of an entire genom cloned into vectors
cDNA library
Fragments prepared from total DNA of cell or tissue and comprises copies of mRNA
Polymerase chain reaction (PCR)
Denaturation, annealing, elongation
Tools for PCR
dNTP, primer, Dna polymerase(thermostable), template
Blotting teqniques
RNA-nothern
DNA-soutern
protein-western
STR
short tandem repeat
VNTR
variable number of tandem repeats
SNP
single nucleotide polymorphism
Variation in a single nucleotide that occurs at a specific position in a genome
Viral vectors
Retroviral vectors Adenosine deaminase deficiency (ADA) Severe combined immunodeficiency (SCID) Adenoviral vectors Adeno-associated viruses (AAV)
Non-viral vectors
Liposome-based
Modified transposons
Two Enzymes involved in cloning vectors
- Restriction Enzymes
- T4 DNA Ligase
LTR
Enhancer, promoter, transcription initiation (capping), transcription terminator and polyadenylation signal. Expression directed by the viral LTR signals is carried out entirely by host cell enzymes (RNA pol II, poly A synthetase, guanyl transferase).
Retionblastoma
tumosupressor
ASA-PCR
4 primers, added to 3´-end closest to mutation
DNA protected from digestion by
metylation catalyzed by DNA methylase
DNA ligase
catalyzes formation of phosphodiester bond with ATP as cofactor
Plasmid
circular DNA
own replication
5000–>400 000 bp
resistant to antibiotics
has ori
covalentely bonded to bacterial chromosom
Naked dna and do not code genes necessary to encase the genetic material for transfer to a new host
Fingerprinting
based on swquence polymorphism
focuses on differences in lengths of short tandem repeats (STR)
FISH
fluorescence in situ hybridization, used to map location of specific DNA sequences witin fixed nuvlei
CRISPER
DNA gene editing regulatory system. Providing resistance to external genes from bacteriophage. It combines with The Cas and nuclease to target and cleave the DNA of invading phage.
Gene deletions
gene editing
modulation of gene transcription
Cosmid
A plasmid into wich DNA from bacteriophage lambda have been inserted, permits DNA packing in vitro.
vector
A plasmid or bachteriophage where foreign DNA can be inserted for purpose of cloning
RNA-inducable interferon
Provide same protection against RNA lige restriction enzymes.
restriction enzymes
Only present in cells that have enzyme that methylates host DNA–>making it unsuitable substrate for digestion
Site-specific DNA methylases
This enzyme and other exist in pairs
Sticky ends
usefull for constructing hybrid or chimeric DNA
MAy reconnect with thelselves, no net gain of DNA
Blunt ends
can be ligated directley,
ligation is not directional
Homopolymer tailing
A process how Dna anneal with vector. Poly G added to 3´-end of vector and poly d added to 3´endof foreign dna using enzyme terminal transferase.
Direct blunt end ligation
bacteriophage T4 DNA ligase and can join any pairs of ends
Cohesive ends
can be added through PCR amplification
Clone
large population of identical molecules arising from common ancestor
Chimeric or hybrid DNA can be constructed in cloning vectors
