Genome projects and gene tech Flashcards
genome definition
complete set of genes in a cell
what does ‘genome sequencing mean’
to know the exact sequence of bases that make up the entire DNA of an organism
Proteome definition
the full range of proteins produced by cells
Recombinant DNA definition
a cell having two or more sources of DNA
Features of genetic code
universal
degenerate
non-overlapping
5 steps in recombinant DNA tech
- isolation of genes
- insertion
- transformation
- identification
- growth
how can you obtain fragments of DNA
-mRNA –> cDNA via reverse transcriptase
-restriction enzymes
-gene machine
How can we use reverse transcriptase to create DNA fragments
-free DNA nucleotides bind to single stranded mRNA template via complementary BP
-Reverse transcriptase joins DNA nucleotides together to form single stranded cDNA molecules
-DNA polymerase required to make cDNA double stranded
Advantages of using reverse transcriptase
-mRNA is much easier to obtain
-Bacterial DNA doesn’t contain introns as bacteria don’t have enzymes needed for splicing
-To copy a gene which codes for a certain protein, mRNA can be isolated from the cytoplasm of cell types which produce the protein in large amounts
How can we use restriction endonucleases to create DNA fragments
-Restriction endonucleases hydrolyse DNA at specific base sequences
-These are usually either side of a desired gene
-These recognition sequences are often palindromic = base pair read is the same in opposite directions
-The DNA sample is incubated with the specific restriction endonucleases which hydrolyses the DNA into fragments wherever the recognition sequence appears
-If the target gene has recognition sequences before and after the target gene, the fragments will contain the desired gene
What can we add to DNA fragments
promoter regions = allow transcription factors to bind, allowing gene to be expressed
terminator region = cause transcription of gene in interest to stop
Gene machine process to create DNA fragments
-Desired nucleotide sequence fed into a computer
-Synthesis of oligonucleotides (short sequences of nucleotides)
-Assembly of gene = oligonucleotides are overlapped then joined together and made double stranded using PCR
-Gene is inserted into a bacterial plasmid
Advantages of Gene machine
DNA without introns
Artificial genes are easily transcribed and translated by prokaryotes, as they have no introns in their DNA
Insertion of genes into a vector process
-Isolated Target DNA fragment inserted into the vector DNA by cutting open the vector DNA using the SAME restriction endonuclease that was used to isolate the DNA fragment
-produces complementary sticky ends between the ends of DNA fragment and cut ends of vector DNA
-Target DNA fragment anneals to vector DNA by complementary base pairing between their sticky ends
-DNA ligase is used to join the DNA fragment and vector DNA at the sugar phosphate backbone by forming phosphodiester bonds.
-The combined DNA fragment and vector DNA is recombinant DNA
Vector definition
A vector is a DNA carrier eg. virus used to transfer foreign DNA into cells
Transformation producing recombinant organisms process
- make artificial DNA with correct sequence of bases
- using DNA polymerase
- cut plasmid open
- with same restriction endonuclease
- sticky ends attached;
- use DNA ligase to join
- return plasmid to bacterial cells
Why must transformed cells be identified
- Not all vectors take up target DNA to become recombinant
- Not all host cells become transformed, by taking up recombinant vectors
Cloning via bacteria
-Once you have identified the transformed bacterial cells then you can culture these bacteria as they reproduce by binary fission to produce genetically identical cells with the desired gene
-These organisms will produce large quantities of the desired protein
cloning via PCR
- Heat DNA to 95 degrees to break weak hydrogen bonds
- Add primers and add nucleotides
- Cool to 55 degrees to allow the binding of nucleotides and primers
- Add thermostable DNA (Taq) polymerase
- Heat to 75 degrees
- DNA polymerase joins nucleotides together
- Repeat cycle many times
Why is PCR more appropriate than cloning via bacteria
Quicker so can produce millions of copies of target DNA within hours
How can we use viruses as vectors in gene therapy
-If foreign DNA fragments are introduced to viral genetic material, it will insert the foreign gene at the same time as its own genetic material
-Viral DNA is cut using the same restriction endonuclease and joined to foreign DNA using DNA ligase
-The virus then acts as a vector
However
-Viruses can cause an immune response and the formation of cytotoxic T cells and memory B cells
-The next time the virus is given, the secondary response is stimulated, and antibodies produced
-To combat this, vector viruses are further modified to evade the immune response
How can we use liposomes as vectors in gene therapy
-Liposomes are lipid droplets which can cross the phospholipid bilayer and releases target DNA into the cell
However
-the DNA does not move into the nucleus and so does integrate into the genome of stem cells lining the airways
-so new daughter cells will not have the functional gen
-from this DNA, small amounts of mRNA can be produced
what is somatic gene therapy
DNA transfer to our normal body tissue
what is germline gene therapy
DNA transfer to cells that produce eggs or sperm
limitations of germline therapy
-imperfect due t random fusion of gametes
-denial of human rights
-used to eliminate disease but also enhance favourable characteristics
limitations of somatic gene therapy
-not all cells take up new DNA
-not all cells express DNA allele
-body can produce an immune response to the vector
Processing of genetic screening using DNA probes and DNA hybridisation
- sequence target gene
- create DNA fragments of complementary target gene
- conduct PCR to obtain large sample of DNA fragments
- make probe = DNA fragments + marker
- target DNA strand separated by heating then cooled with probe
- probe binds to target ssDNA
- washed to remove excess probe
- hybridised DNA observed through special microscope
what is DNA probe
-A short, single stranded DNA molecule with a complementary base sequence to DNA fragment to be located which is radioactive or labelled by a fluorescent molecule
What does VNTR mean?
Variable Number Tandem Repeats
Process of DNA fingerprinting
- DNA extracted from sample
- DNA hydrolysed into segments by restriction endonucleases
- VNTR’s left intact
- DNA fragments separated using electrophoresis
- Mixture put into wells on gel and electric current passed through
- Immerse gel in alkaline solution to separate DNA strands
- Southern blotting=cover with nylon to absorb DNA
- DNA fixed to nylon membrane by UV light
- Radioactive marker with probe added which hybridises to target fragments
- areas with probe bidentified using X-ray film
