Unit 3 - Biotechnolgy pt 1 (manipulating and cloning DNA) Flashcards
Genetic Engineering
the intentional production of new genes and alteration of genomes by the substitution or introduction of new genetic materil
ie. human insulin
Genetic engineering example
- human gene that code for insulin is inserted into EE.coli plasmid
- E.coli transcribes and translates the piece of human DNA to make the human protein insulin which is then harvested from the bacteria (safflower)
Recombinant DNA
a DNA strand created using DNA pieces from two or more sources
Restriction Enzymes
act like molecular scissors and cut DNA at specific locations and recognizes a specific sequence of nucleotides on DNA strand
recognition site
these r the specific location that r 4 to 8 pairs long and are palindromic (both strands)
Restriction fragment
are the fragment produced when a DNA strand is cut by a restriction enzyme
Restriction enzyme
each enzyme is cut at only one recognition site and in only one direction
types of ends
blunt and sticky
blunt
is cut made straight across the strand
sticky
cut in a zigzag. scientist prefer to work with sticky ends since they are easier to rejoin w other fragments cut with the same enzyme
History of Restriction enzymes
Dr. Hamiliton smith of johns hopkins discovered them in 1970s
since then 2500 have be catalogued with about 200 diff target sequences
Restriction enzymes naming process
named after its cell origin plus a roman numeral if more than one restriction enzyme has been isolated from species
DNA ligase
is the enzyme that is used to join cut strans of DNA
T4 DNA ligase
works well with blunt ends
-less efficient more likely to fail
Methylases
enzymes which add a methyl group to adenine to cytosine base within the recognition sequence preventing enzyme from hydrolyzing cells own DNA
competent cell
is a cell that is able to take up foreign DNA from surroudings
Host cell
a cell that has taken up a foreign plasmid or virus and has used its cellular machinery to express foreign DNA
Vector
DNA mol used as a vehicle to transfer foreign genetic material into cell
ie. plasmid
cloned gene
and identical copy of an original target gene that can be made by introducing the target gene into a host cell and having a copie
Plasmids
common vector used for transferring foreign genetic material into a cell
Restriction map
a diagram that shows the relative location of all the know restriction enzyme recognition sites on a particular plasmin and the distance, in to between the sites ( helps pick the correct plasmid to use a host for recombinant DNA procedure)
Transformation
under the specific conditions, plasmid enter bacterial cells multiply and express the foreign gene that has been inserted into the plasmid genome
DNA cloning
occurs after the use of restriction enzymes and ligation and transfers newly made plasmids to bacteria. after tranformation, bacteria r selected on antibiotic plates. bacteria with a plasmid r antibiotic forming a colony
colonies
with the right plasmid can be grown to make large cultures of identical bacteria which r used to produce plasmin or make protein
Transformation; some bacteria don’t take up plasmids naturally…
- instead these bacteria r placed in solution w calcium chloride and recombidiant DNA plasmids
heat shock
through this process of heating and cooling the bacteria cell membrane is disrupted and the plasmid enters
steps of bacterial transformation (1)
specially prepared bacteria r mixed w recombinant DNA filled plasmids
steps of bacterial transformation (2)
bacteria r given a heat shock which causes some of them to take up plasmid
steps of bacterial transformation (3)
plasmids used in cloning contain an antibiotic resistance gene. thus all of the bacteria r placed on an antibiotic plate
steps of bacterial transformation (4)
bacteria without a plasmid die. each bacterium with a plasmid gives rise to a cluster of identical plasmid containing a colony
steps of bacterial transformation (5)
several colonies check to identify one w the right plasmid (PCR)
steps of bacterial transformation (6)
a colony containing the right plasmid grown in bulk and used for plasmid or protein production
