21: Recombinant DNA Tech Flashcards
What is recombinant DNA?
DNA of two different organisms that has been combined
What is an organism with recombinant DNA inserted called?
Transgenic
Genetically modified organism (GMO)
Why can recombinant DNA be inserted into an organism?
Genetic code is universal
What is the process of making a protein using DNA technology of gene transfer?
Isolation Insertion Transformation Identification Growth/cloning
What occurs in isolation?
DNA fragments for desired gene are isolated
What occurs in insertion?
DNA fragment collected is inserted into a vector
What occurs in transformation?
Transfer of DNA into suitable host cells
What occurs in identification?
Host cells which have taken up the gene is identified using gene markers
What occurs in growth/cloning?
The host cells population is replicated/cloned to produce many copies
What are the methods of producing DNA fragments?
Conversion of mRNA to cDNA using reverse transcriptase
Restriction endonucleases used to cut fragments containing desired gene from DNA
Creating a gene in the gene machine, from a known protein structure
What is cDNA?
Complementary DNA
As made of nucleotides which are complementary to the mRNA found
What is the process of using reverse transcriptase to make DNA fragments?
Cell which produces the protein is selected as have large amounts of relevant mRNA
Reverse transcriptase converts mRNA to ds cDNA
cDNA made to be ss by hydrolysis with an enzyme
DNA polymerase enzyme builds complementary nucleotides on cDNA template
dsDNA is the required gene
What are restriction endonucleases?
Enzymes found in bacteria which cut up viral DNA
Viruses inject DNA into bacteria to take over cell
What is the difference between different restriction endonucleases?
Each one cuts a dsDNA at a specific base sequence
What is a recognition sequence?
Specific base sequence of DNA at which a restriction endonuclease cuts
What are blunt ends of DNA?
Restriction endonuclease cuts DNA between two opposite base pairs
Leaves two straight edges called blunt ends
What are sticky ends of DNA?
Restriction endonuclease cuts DNA in a staggered fashion
Leaves an uneven cut in which each strand of DNA has exposed, unpaired bases
What is a palindromic sequence?
The unpaired bases read from left to right is the same as right to left
What is the gene machine?
A piece of equipment which manufactures genes in a laboratory
How does a gene machine work?
Desired nucleotide sequence of genes determined from desired protein
Desired sequence is fed into the computer
Sequence checked for biosafety and biosecurity to keep to international and ethical standards
Computer designs series of oligonucleotides which can be assembled into a desired genes
Automated process, each of oligonucleotides assembled one nucleotide at a time
Oligonucleotides joined together to make a gene, replicated using PCR
What is an oligonucleotide?
Small, overlapping single strands of nucleotides
What is the feature of the gene made from a gene machine?
No introns or other non-coding DNA
What is done with genes made from the gene machine?
Sticky ends used to insert gene into a bacterial plasmid (vector)
Then can be used to store, clone or transfer to different organism
What are the advantages of the gene machine?
Any nucleotide sequence can be produced in a short time with great accuracy
Artificial genes are free of introns so can be transcribed & translated by prokaryotic cells
What is a vector?
Carrying unit for the DNA fragment
E.g. bacterial plasmid
How can cloning of a gene be done?
In vivo - transfer to a host cell using a vector
In vitro - using PCR
How are sticky ends used in vectors?
Exposed bases made from sticky ends
Complementary base pairing occurs between this and DNA from another source cut with same restriction endonuclease
DNA ligase joins the two sections
What does DNA ligase do?
Forms the sugar-phosphate backbone between the two different sections of DNA
What is a promoter?
Region of DNA which transcription factors and RNA polymerase binds to, allows transcription to occur
What must be done to a DNA fragment before it is inserted?
Promoter and terminator region has to be added to the DNA fragment
Why do promoter and terminator regions be added to DNA fragment?
Ensures that the DNA is transcribed and made when it is inserted into a living organism
What is the most commonly used vector?
Plasmid
What are plasmids?
Circular lengths of DNA found in bacteria
Separate from main bacterial DNA
Contain genes from antibiotic resistance
Why is the restriction endonuclease used for the DNA the same as the one used for the plasmid?
Ensures sticky ends of opened-up plasmid are complementary to sticky ends of DNA fragment
What is transformation?
Process of plasmid being inserted into the bacterial cells
What is the process of transformation?
Plasmids and bacteria mixed in medium with Ca2+
Ca2+ and changes to temp makes the bacterial membrane permeable
Plasmids move into the cytoplasm through the membrane
Why do all of the bacterial cells not have the DNA fragments?
V few bacterial cells take up plasmids when mixed
Some plasmids will have closed up again without incorporating the DNA fragment
Sometimes DNA fragment ends join back together to form own plasmid
How many genes can a plasmid carry?
Many genes for resistance
One plasmid can dictate resistance from multiple antibiotics
What is a marker gene?
A gene used to determine if a fragment has been successfully inserted into an organism’s DNA
What are some examples of marker genes?
Antibiotic-resistant genes
Fluorescent protein producing gene
Enzyme producing gene whose action can be identified
Is antibiotic-resistant marker genes used?
Old technology
Superseded by other methods
How is resistance for ampicillin used to find which bacterial cells have taken up the plasmid?
All bacteria cells grown on medium with ampicillin
Bacterial cells with plasmids will be resistant to ampicillin
Bacterial cells break down ampicillin
Others do not take up plasmid and therefore die
What is replica plating?
Process used to find those cells with plasmids that have taken up the new gene
What is the process of replica plating?
Filter paper/ felt used to obtain an exact copy of the colony locations on the original plate
Used to transfer to plate containing antibiotic (ampicillin) which all with plasmids have
Those that are alive are used to transfer colonies to plate with different antibiotic (tetracycline) which the fragment would be inserted into the middle of
Those which die have the correct plasmid, those which live have the unaltered plasmid
Replica plating means colony with recombinant DNA can be found on ampicillin plate