gene tech Flashcards
what is a transgenic organism?
organism that contains recombinant DNA/ DNA of a different species.
what is recombinant dna?
DNA of 2 different species which has been combined
why can a gene from one species be transplanted into gene from another species and still get same protein?
the genetic code is universal (each triplet code, codes for the same amino acid in ALL organisms) and the transcription and translation mechanisms are universal
what are the 5 steps in making recombinant dna with in vivo gene cloning?
- Isolation of the gene/DNA fragment for the desired protein
- Insertion of the DNA fragment into a vector
- Transformation – Putting DNA fragment into host cells
- Identification of host cells which have taken up the gene
- Cloning/growth of these cells
describe isolation of a dna fragment (step 1) in making recombinant dna
i. Conversion of mRNA to complementary DNA (cDNA) using reverse transcriptase: mRNA coding for the desired protein homogenised + centrifuged (e.g. for insulin, the mRNA from pancreatic β cells).
· Free DNA nucleotides are added and bind to single stranded mRNA via complementary base pairing + reverse transcriptase is added which joins adjacent DNA nucleotides together via phosphodiester bonds.
· This forms a single stranded DNA called cDNA + more nucleotides and polymerase added to make it single stranded.
Advantages: introns removed so prokaryotes can use + easy to remove bc outside of nucleus.
ii. Using restriction enzymes: Restriction enzymes hydrolyse the phosphodiester bonds of DNA at specific base sequences called recognition sites. Different restriction enzymes cut DNA at different specific recognition sites because the shape of the recognition site is complementary to the enzymes active site.
The cut made at the recognition site produces sticky ends. If the other organism’s DNA is cut using the same restriction enzyme as they will have complementary sticky ends, so hydrogen bond together.
iii. Using a gene machine: faster bc don’t need to isolate DNA/RNA, very accurate like no errors, any nucleotide sequence, no introns so prokaryotes can use.
describe insertion of dna into a vector for making recombinant dna (step 2)
A vector (i.e. bacterial plasmid or virus) carries the DNA/gene into the host cell.
The donor gene need a promotor + terminator, so gene can be transcribed + stop transcription.
· The isolated DNA fragment is placed into vector DNA by cutting open the vector DNA using the SAME restriction enzyme that was used to isolate the DNA fragment, producing complementary ‘sticky ends’ between the DNA fragment and vector DNA, which join via CBP at their sticky ends.
· DNA ligase is then used to join the phosphodiester bonds of the sticky ends of the DNA fragment to the vector DNA (e.g. plasmid).
· The new combined DNA fragment and vector DNA is referred to as recombinant DNA.
describe transformation of dna fragment in making recombinant dna (step 3)
recomb DNA now used to transfer target DNA into host cell by:
* Calcium ions OR electric Shock OR heat shock (these make the membrane porous and therefore more permeable)
* Using a virus to inject the plasmid/DNA into the cell
organism with the recombinant dna = trasngenic
describe identification of host cells which have taken up gene in vivo gene cloning (step 4)
Marker genes used: identifies which cells have taken up the gene.
· Antibiotic resistance gene – marker gene codes for a protein which gives resistance to a certain antibiotic. Cells are grown in the presence of this certain antibiotic and therefore only the cells containing the vector with the antibiotic resistance gene will grow. Cell without the vector will not have the antibiotic resistance gene and therefore will die.
· Fluorescence gene – marker gene codes for a fluorescent protein. When UV light is shone on cells, only the cells which have taken up the plasmid and produce the fluorescent protein will glow.
describe cloning/ growth of host cells which have taken up recombinant dna (step 5)
Once the cells containing the recombinant DNA have been identified they can be cultured to express the inserted gene and therefore produce large amounts of the desired protein.
· Gene can be inserted into early embryo or egg cells.
All resulting cells in the organism will produce the protein because the cells divide by mitosis to produce genetically identical cells
what is PCR?
Polymerase chain reaction (PCR) is used to amplify (make many copies) of a particular DNA sequence from a small sample- much more rapid + efficient than in vivo.
what are primers?
Primers are short pieces of single stranded DNA with a specific, complementary base sequence to bases at start of DNA fragment you want to copy.
describe PCR xxxx
A reaction mixture is set up containing: the DNA sample to be copied, free DNA nucleotides, 2 different primers (one for each strand which have different base sequences) and thermostable DNA polymerase.
· DNA is heated to 95oC to break hydrogen bonds and separate the two DNA strands. (1)
· Cooled to 55oC so that complementary primers can bind to DNA strands, allowing DNA polymerase to bind and prevent the strands re-joining. (2)
· Heated to 72oC - Thermostable DNA polymerase joins DNA nucleotides together via phosphodiester bonds forming a new complementary strand. (3)
· Two new copies of the DNA fragment are formed
· This cycle can be repeated many times - number of copies of DNA fragments doubles with each cycle leading to an exponential increase. (4)
· No more DNA molecules are produced once the nucleotides or primers have all been used up.
how to calculate number of dna molecules produced from PCR
2(to the power of)n x number of starting molecules - where n is the number of cycles.
state 3 differences between in vivo VS in vitro (PCR)
in vivo:
1) Can be used to produce protein or mRNA from the inserted DNA as well as the target DNA.
2) Not very sensitive – need large DNA sample to start with.
3) Slower – One DNA replication per cell division
in vitro:
1) Can only be used to copy DNA
2) Very sensitive – only requires a small DNA sample to start with.
3) Quicker –millions of copies of target DNA in hours.
how can gene therapy treat recessive + dominant alleles which cause disease?
- If the disease is caused by a recessive allele then a dominant allele can be introduced – which will be expressed instead of the recessive allele.
- If the disease is caused by a dominant allele then the functional gene can be inserted into the middle of the mutated allele, disrupting it, so the functional protein is produced instead.
