Paper 2 - Gene Tech And Genome Sequencing Flashcards
Producing DNA Fragments - Restriction endonucleases
These recognise and cut DNA at a specific sequence of bases, creating sticky ends. These are palindromic - also complimentary when reversed
Producing DNA Fragments - Reverse Transcriptase
This converts mRNA to cDNA , making it easier to isolate mRNA and the cDNA will have introns removed
What are retroviruses ?
Viruses that contain RNA, not DNA.
Producing DNA Fragments - Using a gene machine
- Amino acid sequence of the protein is determined
- The mRNA codons are looked up
- The complementary DNA triplets are worked out = gene produced
- Gene is produced by creating many short fragments and joining them together
- Genes are checked and those with errors are rejected
From this any sequence of nucleotides can be produced in a short period with great accuracy. Genes would also be free of introns.
Polymerase Chain Reaction (PCR)
- DNA polymerase, free nucleotides and primers are in a mixture.
- Primers’ specific base sequence is complementary to the ends of the fragments. - DNA Polymerase can bind
- Temperature increased to 95 degrees, breaking hydrogen bonds between bases to separate the strands of DNA.
- Each strand can act as a template to build new complementary strands.
- Temperature decreased to 40 degrees. = primers attach to complementary base sequences at ends of single strands
- Temp increased to 72 degrees and DNA polymerase attaches = free nucleotides are joined to primer with phosphodiester bonds
- Cycle repeats and DNA is doubled in quantity each time
Define genome
All the genes in an organism
Define proteome
The range of different proteins that an organism codes for
What is recombinant DNA ?
DNA formed by joining together DNA from 2 different organisms
Insertion of DNA fragments into a vector
- A restriction enzyme is used to cut the gene with sticky ends. Promotor and terminator regions are added.
- Same restriction enzyme cuts the plasmid
- Gene and plasmid are mixed and are joined together due them having complimentary sticky ends
- Fragments are joined by DNA ligaments = recombinant plasmid
How can bacterial cells be transformed ?
- Plasmids and bacterial cells are mixed in ice cold calcium chloride, heat shock = cell walls more permeable
- Plasmids are introduced into bacterial cells
- Cells that contain the recombinant DNA = transformed organisms
Some cells may contain original plasmid or have circularised DNA fragments so will not be transformed.
Somatic gene therapy
This alters alleles in a person’s body cells - offspring could still inherit the disease
Germ-line therapy
This alters alleles in sex cells - no offspring will suffer the disease
Genetic fingerprinting
- Extract a sample of DNA and cut using restriction enzyme to produce DNA fragments
- Separate fragments according to size using gel electrophoresis
- Transfer to nylon membrane to make fragments single stranded
- Apply radioactively labelled DNA probe which is complimentary to the VNTR’s in the fragments
- Use X-ray film to visualise position of DNA fragments
- Pattern of bands is unique to every individual (except twins)
Gel electrophoresis
- DNA sample is loaded into a well in an agar gel plate
- An electrical current is passed through the gel
- DNA is attracted to the positive electrode due to negatively charged phosphate group
- Molecules diffuse through the gel towards the positive electrode
- The DNA fragments are separated according to mass/length - shorter lengths of DNA move faster and travel further
Medical diagnosis
- Extract a sample of DNA from patient and cut using restriction enzyme to produce DNA fragments
- Separate fragments according to size using gel electrophoresis
- Transfer nylon membrane to make fragments single stranded
- Apply flurorescently labelled DNA probe which is complimentary to the gene/allele
- Wash to remove unbound probe
- Use UV light to show any bound probe
- If fluorescence seen = patient has gene
- Double the amount of fluorescence = 2x allele (homozygous)
