Chapter 21- Recombinant DNA technology Flashcards
What is recombinant DNA
Where fragments of foreign DNA is inserted into other sections of DNA
What is a transgenic organism
An organism that can successfully express a gene from any organism
What are the 3 methods used to produce DNA fragments
- conversion of mRNA to cDNA using reverse transcriptase
-using restriction endonucleases to cut fragments containing the desired gene from DNA
-creating the gene in a known gene machine usually based on a known protein structure
How is reverse transcriptase used to create DNA fragments
- The cell that readily produces the protein is selected
- These cells have large quantaties of the relevant mRNA which is therefore more easily extracted
- Reverse transcriptase is then used to make DNA from RNA. This DNA is known as complementary DNA (cDNA) because it is made up of nucleotides complementary to the mRNA
- DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand is the required DNA.
What are restriction endonucleases
enzymes, extracted from bacteria, that cut DNA at specific palindrome sequences to isolate DNA fragments
What are palindrome sequences
sections of DNA where the nucleotide sequence reads the same in opposite directions on antiparallel strands.
how are restriction endonucleases used to cut DNA
DNA is incubated with chosen restriction enzyme(s).
Restriction enzymes identify palindromic sequences in the DNA double helix and cut double-stranded DNA if their recognition sequence is present.
Recognition sequences at either end of a desired DNA fragment allow restriction enzymes to separate the fragment from the rest of the DNA to obtain the desired gene.
Enzymes cut target gene fragment out via hydrolysis reaction.
Different restriction enzymes cut at different sequences based on their active site shape.
How are gene machine used to create new DNA sequences
Choose codons for the desired amino acid sequence from a known protein structure.
Use a computer to direct the synthesis of short fragments of DNA (oligonucleotides) in gene machines.
Join the fragments together to make a longer sequence of nucleotides, forming the desired gene.
Polymerase chain reaction (PCR) constructs a complementary DNA strand and amplifies the gene to produce multiple copies.
what is in vivo gene cloning
by transferring fragments to a host cell using a vector. Used to amplify DNA fragments
what is in vitro gene cloning
using the polymerase chain reaction used to amplify dna fragments
How is the DNA prepared for insertion
- promotor and terminator regions need to be added to DNA fragments before they can be inserted
- promotor regions provide a binding area for RNA polymerase and transcription factors to begin the process of transcription
- terminator regions release ran polymerase to end transcription
How are DNA fragments inserted into a vector
- Plasmid cut open by same restriction endonuclease
- this creates the same sticky ends
- DNA fragment sticky ends are complementary to the sticky ends on the plasmid
- DNA fragment and plasmid are combined with DNA ligase which anneals them together by catalysing the condensation reaction to form phosphodiester bonds between nucleotides
How is DNA inserted into bacterial cells
- bacterial cells and plasmids mixed together in a medium containing calcium ions and are heat shocked
- calcium ions make the bacterial membrane permeable allowing plasmids to pass through the cell-surface membrane into the cytoplasm
Why won’t all bacterial cells take up the plasmid
- The recombinant plasmid doesn’t get inside the cell
- The plasmid re joins before the DNA fragment enters
- The DNA fragment sticks to itself rather than inserting into the plasmid
What are marker genes used for
To identify which bacteria successfully took up the plasmids
What are the 3 types of marker genes used
- Antibiotic resistance genes
- Genes coding for fluroescent proteins
- Genes coding for enzymes
How are antibiotic resistance marker genes used
-Tetracycline resistance gene will have been cut in order to insert the DNA fragment
-plasmid will no longer produce the enzyme to break down tetracycline
- can identify these bacteria by growing them in a culture of tetracycline
- the plasmids that has successfully been taken up will die as they do not contain the gene that codes for the enzyme to break down tetracycline
How are fluorescent markers used
jellyfish contain a gene which codes to create a green fluorescent protein (GFP)
this GFP can be inserted into the bacteria plasmid
DNA fragment is inserted into the middle of the GFP gene
This disrupts it and prevents GFP production
Bacteria grown on agar
Only non-glowing colonies contain the DNA fragment
How are enzyme markers used
- lactase can turn a certain substrate from colourless to blue
- lactase inserted into plasmid
- DNA fragment inserted into the middle of lactase gene to disrupt it
- bacteria grown in agar containing substrate
- colonies which cannot turn the substrate blue contain the recombinant plasmid
How is the host cell grown
- a fermenter is used to grow multiple copies of the host cell which have been identified as containing the recombinant plasmid
- this large, cloned population of the host cell can then produce the protein coded for by the inserted DNA fragment
What is the polymerase chain reaction?
a method of copying fragments of DNA. The process is automated so it is rapid and efficient
What are primers
short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments
What is a thermocycler
a computer controlled machine that varies temperatures precisely over a period of time
