Chapter 7: 7.1 Basics of Genetic Engineering Flashcards
What does genetic engineering allow us to identify?
THe role of every amino acid in a protein
What is genetic engineering used to study?
Protein structure and function
Genetic manipulation
Select genes can be input (“cloned”) into plasmids either in their native form or manipulated/mutated form for genetic manipulation or protein analysis
Genes can be cloned from any ——- DNA
Genomic
Can cloned genes be manipulated?
Yes
What are the 4 steps in cloning?
- Clone gene
- Insert cloned gene into plasmid
- Insert plasmid into host
- Grow host
Cloning:
What can the gene be cloned from?
- cDNA
- mRNA
- Genomic DNA (prokaryotes only)
What are expression vectors in cloning?
Plasmids
Plasmids
- Circular form of dsDNA
- Often are/are derived from naturally occuring bacterial DNA
The insertion of a gene of interest into plasmid results in…
Recombinant DNA
* DNA consisting of genetic material from several sources
What are the key components of plasmids?
- Origin of replication
- Selection marker
- Multiple cloning site
- Promoter
What are circularized plasmids replicated in bacteria for?
To produce more:
* Plasmid
* Protein (translated from gene insert)
Restriction enzymes
Restriction endonuclease (RE)
* Cut dsDNA at palindromic sites
What did restriction endonucleases come from?
Prokaryotic cells
What types of ends can restriction enzymes create?
- Blunt ends
- Sticky ends
What must be introduced to the ends of your gene of interest if you want to insert it into a plasmid?
Introduce RE sites to ends of gene
* Done by PCR
The RE sites at the end of your gene of interest must match…
Sites in the plasmid of interest
After RE cuts the gene of interest and the plasmid, what fuses them together?
DNA ligase fuses the complementary sequences together
PCR
Polymerase Chain Reaction
* Used to amplify and alter genetic material using short complementary oligos called primers
What are the steps in PCR?
- Denaturation
- Annealing
- Elongation
- Repeat
Steps in PCR:
Denaturation
Double-stranded DNA is separated at high temperatures
Steps in PCR:
Annealing
Primers bind to their complementary DNA at primer specific temperatures
Steps in PCR:
Elongation
Nucleotide bases are added onto the ends of primers by thermostable DNA polymerase
In PCR, how many genes are present after each cycle?
Double the number of genes
