Recomb. Tech
joining together of DNA molecules from two or more different species that are inserted into a host organism to produce new genetic combinations.
What is Recombinant DNA Technology?
Mixture temp. raised to 72*C & kept at a set of time for DNA poly to elongate each primer by copying the single-stranded template.
What is PCR Extension?
at least 107 copies of target DNA may be produced by means of this thermal cycling.
What is PCR 25 to 30 cycles?
DNA isolation/Gene synthesis & PCR
What is the first step of Recombinant DNA Tech?
Introduction/insertion of the ligand plasmid into host cells generating a mixed population of transformed and non-transformed host cells.
What is Transformation?
small double-stranded DNA molecules that can exist independently of host chromosome.
What is Plasmids?
Mixture is rapidly cooled to defined temp. (specific to primer) allowing 2 primers to bind to sequences on each of the 2 flanking strands on target DNA.
What is PCR Primer Annealing?
(1) Denaturation, (2) Primer Annealing, (3) Extension
What are steps of PCR?
A suitable plasmid is isolated from a bacterium.
What is the second step of Recombinant DNA Tech?
filtering the transformed host cells only.
What is Selection Process?
Origin of replication (ORI), Promoter & terminator, Ribosome binding site (RBS), Gene of interest (ATG --> STOP codon), Selection marker (antibiotic resistance gene)
What are essential components of an expression vector (plasmid)?
Reaction mix heated to 95*C for (15-30 sec) to break H-bonds between base pairs in template DNA --> single-strands (act as templates for DNA synthesis).
What is PCR Denaturation?
DNA fragment containing gene of interest is obtained.
What is DNA isolation/Gene synthesis & PCR?
The DNA fragment containing the gene of interest and the plasmid are cut by the same restriction enzymes (lacZ).
What is the third step of Recombinant DNA Tech?
Marker gene of plasmid is employed (e.g. AmpR marker).
What is isolation of recomb. cells from non-recomb. cells?
ColE1, P15A, SC101 (Organism Specific)
What is ORI?
Addition of nucleotides to 3' terminus end of each annealed primer and extension of sequence complementary to target template by DNA poly.
What is PCR Extension?
colony of bacteria that grows on the selective plate (it took up a plasmid with the antibiotic resistance marker) but does not contain the correct recombinant DNA (the vector + insert). Instead, it contains a different, undesired DNA molecule.
What is a False Positive Colony?
The DNA fragment containing the gene of interest is inserted into the plasmid by ligase.
What is the fourth step of Recombinant DNA Tech?
Non-specific PCR product, self-ligation, incorrect ligation, contamination.
What are reasons for false positive colonies?
AmpR, KanR, CmR, TetR (Antibiotic Resistance)
What is Selection Marker?
both double-stranded products of cycle 1 are denatured and subsequently serve as targets for more primer annealing and extension by DNA poly.
What is PCR cycle 2?
Multiple inserts ligating together into the vector; Vector re-ligation; Insert-only ligation or tandem repeats of the insert.
What is Incorrect Ligation?
Verification of the correct recombinant plasmid construct.
What is the fifth step in Recombinant DNA Tech?
Restriction enzyme digestion, colony PCR, DNA sequencing.
What are Verification methods?