Unit 6 - Gene technologies Flashcards
Definition of a gene?
A DNA base sequence which codes for a polypeptide or functional RNA.
Definition of genome?
The complete set of genes in a cell.
Definition of proteome?
The full range of proteins that a cell is able to produce.
What do genome projects do?
They determine the DNA base sequence of an organisms genome. This allows the amino acid sequences of the proteins to be determined (the proteome).
What is it easier to determine the proteome of prokaryotic DNA?
It has no introns, is shorter so less genes, DNA is only 1 piece of circular DNA and it is not associated with histone proteins.
In eukaryotes, the introns and regulatory genes mean that the genome cannot easily be translated into the proteome.
What is a use of identifying the proteome of microorganisms?
It allows the identification of antigens for use in vaccine production.
What does In Vivo stand for,
In a living organism.
What is the definition of recombinant DNA?
DNA made from 2 or more different species.
In vivo gene cloning: what are the 3 methods for isolating the gene and producing a DNA fragment?
1) Reverse transcriptase
2) Restriction enzymes
3) Gene machine
In Vivo gene cloning: How is reverse transcriptase used to isolate the gene?
1) The mRNA coding for the desired protein is extracted from the cells using homogenisation and differential centrifugation.
2) Free DNA nucleotides are added and bind to single stranded mRNA via complementary base pairing. A-U, T-A, C-G, G-C
3) Reverse transcriptase is added which joins adjacent DNA nucleotides together via phosphodiester bonds.
4) This forms a single stranded DNA called cDNA (complementary DNA).
5) The addition of further DNA nucleotides and DNA polymerase is then used to make cDNA double stranded.
In vivo gene cloning: What are the advantages of using reverse transcriptase to isolate a gene?
-Using mRNA from eukaryotes means introns have been removed, so DNA can be used in prokaryotes, as prokaryotes cannot splice pre-mRNA so cannot remove introns.
-mRNA is relatively easy to isolate as it can be found outside of the nucleus.
In vivo gene cloning: How are restriction enzymes used to isolate a gene?
1) Restriction enzymes hydrolyse phosphodiester bonds at recognition sites (specific base sequences) on DNA.
2) These sequences are palindromic, they read the same in opposite directions.
3) They form ‘sticky ends’.
Definition of a sticky end?
It is a short, single stranded section at the end of a DNA molecule.
What are the advantages of using a gene machine?
-Very accurate, no errors
-Faster as no need to isolate DNA or RNA from cell
-No introns, so can be expressed by prokaryotes
-Any sequence of nucleotides can be made
In vivo gene cloning: How can DNA be inserted into a host cell using a plasmid?
A promoter and terminator must be added to the DNA fragment. A plasmid can be used as a vector and can be cut open using the same restriction enzymes as was used to cut out the DNA fragment. The sticky ends on the plasmid are now complementary to the sticky ends on the gene. They hydrogen bond via complementary base pairing. DNA ligand joins the sticky ends of the plasmid and DNA/gene together via phosphodiester bonds. This is now recombinant DNA.
What is a promoter?
They bind to transcription factors for transcription.
What is a terminator?
A region added to the end so transcription can stop.
What is a vector?
It carries the gene into a cell.
In vivo gene cloning: How can you introduce the DNA fragment into a suitable host cell?
There are multiple processes that can be used to make the bacterial host cell take up the plasmid/
-Adding calcium ions
-Electric shock
-Heat shock
These all make the membrane more porous
-Using a virus.
In vivo gene cloning: How can you identify the host cell that has taken up the required gene?
You add a marker gene which identifies which cells have been taken up the plasmid/gene.
How does an antibiotic resistance marker gene identify the bacteria containing the recombinant plasmid?
Only the bacteria containing the plasmid survive. All other bacteria die.
How does replica plating work?
It involves using 2 antibiotic resistant marker genes in the same plasmid. The DNA fragment needs to be inserted into one of these marker genes, disrupting it and preventing it from working. The bacteria colonies are transferred from a master plate to the 2 antibiotic plates, in exactly the same position. The recombinant plasmid DNA will survive in one bacteria plate but not in the other.
