Ch 20 Flashcards
recombinant DNA
DNA molecules formed when segments from different sources are combined in vitro
in vitro
in a test tube
biotechnology
manipulation of organisms or their components to make useful products
genetic engineering
direct manipulation of genes
DNA Cloning
production of copies of defined DNA segments
plasmid
small circular SNA molecules that replicate separately form the bacterial chromosome
Steps of DNA Cloning
- gene inserted into plasmid
- plasmid put into bacterial cell
- host cell reproduces through cell division to form a clone of cells
clone
population of genetically identical cells
gene cloning
production of copies of a single gene
amplify
to make many copies of a gene
What do restriction enzymes do?
cut DNA molecules
restriction/recognition sites
specific sequence where a restriction enzyme can cut DNA into restriction fragments
What is special about a restriction site?
the sequence is a palindrome
sticky end
single-stranded end of fragment where it can attach to others
DNA ligase
enzyme that seals sticky ends together
isoschizomer
different restriction enzymes that produce the same cut
cloning vector
DNA molecule that can carry foreign DNA into a host cell and replicate
Set up for cloning genes in a bacterial plasmid
- Obtain gene of interest and a plasmid with a resistance gene
- Cut with the same restriction enzyme. Plasmid will be cut in the lac z gene.
- Mix plasmids and fragments; some will join.
- DNA mixture added to bacteria
Steps to identify the correct bacterial plasmid
Place bacteria in a medium with ampicillin and x-gal.
Only bacteria with the plasmid will reproduce because they are amp resistant.
Only bacteria containing a recombinant plasmid/insert in lac z will turn white because they can’t hydrolyze x-gal.
cDNA
complementary, double stranded DNA formed form mRNA
Enzyme for cDNA synthesis
reverse transcriptase
cDNA shows only…
expressed genes
Purpose of Nucleic Acid Hybridization
detect specific sequence by its ability to base pair with a complementary DNA
Steps of Nucleic Acid Hybridization
- DNA transferred to a nylon membrane.
- Incubated in a solution of radioactive probe molecules that will base pair with complementary DNA.
- Excess DNA is rinsed off.
- Covered with film that exposes radioactivity
What does PCR stand for?
polymerase chain reaction
purpose of PCR
amplify a sequence through in vitro replication
Steps of PCR
- Denature
- Anneal
- Extend
enzyme of PCR
Taq DNA polymerase to extend and to last through denaturing
Gel electrophoresis
gel separates nucleic acids or proteins by size.
In gel electrophoresis, ________ moves __________.
Shorter moves faster
RFLP
loss of a restriction site = change in band pattern
Southern blotting
detects only bands with part of a specific gene
Steps of southern blotting
- gel electrophoresis
- transfer/blotting to membrane
- hybridization
Northern blotting
same as Southern but mRNA instead of DNA
Steps of Dideoxy chain termination
- Denature strands and anneal primers
- strands synthesized until a dideoxyribonucleotide is added
- Read the tagged nucleotides
