- Start of each gene is marked by sequence of base called a promoter
- RNA is built up by ribose nucleotides which attach themselves to complementary base pairing- only one strand made
- copied strand is called template strand
- New nucleotides joined together by forming phosphodiester bonds by enzyme RNA polymerase
- mRNA molecule peels off from DNA. winding enzymes reminds the DNA
- mRNA diffuses out of the nucleus through nulcear pore.
POST- TRANSCRIPTION MODIFICATION
- Initial mRNA that is transcribed is called pre-mRNA (exact copy of DNA)
- Introns in the mRNA are cut out and exons are joined together bey enzymes in process called splicing
- splicing is done by ribozymes and snurps
- This results in shorter mature RNA containing only exons
- Ribosomes attach to the mRNA at intitation codon (AUG)
- First tRNA molecule with and amino acid attached diffuses to the ribosome.
- Its anticodon attaches to first mRNA codon by complementary base pairing
- Next amino acid- tRNA attaches adjacebt to mRNA codon
- Bond between amino acid and tRNA is cut and peptide bond is formed between two amino acids
- Catalysed by ribozymes
- Ribosomes move along one codon so new one can attach
- Many ribosomes attaches to a sing mRNA called polyribosome or polysome
- Gene mutation due to base pairing error during dna replication
- SUBSTITUTION- only affects one amino acid, if occures in third base it may have no effect as it is degenerate. silent mutation
- DELETION & INSERTION- there will be a frame shift mutation. change in codon reading frame so all amino acid 'downstream' of mutation will be wrong.
- MUTATION RATES AND MUTAGEN
- rate of mutation is incread by chemicals or by radiation, these are called mutagenic agent or mutagens
- high-energy ionising radiation such as Xray Uv ray, alpha and beta.
- intercalating chemicals such as mustard gas
- chemical which reacts with dna such as benzene and tar
MUTATION AND CANCER
- Proto-oncogenes- stimulates cell divisin when activated by growth signals such as hormones. Mutation turns into oncogenes. so cells is to active all the time, mutation is dominant
- Tumour- surpressor genes - Inhibits cell division when activated by stop signal. Mutation cause the gene to stop making protien so cell pass the checkpoint.
- potent- Potential to differentiate inot specialised cell types
- immortal - divide indefinitely
where do they come from
- embryo stem cells- grown invitro from human embryos. These are totipotent- differentiate into any cell type
- adult stem cells - extraction from certain tissues. multipotent- they can differentiate in to their own family cells
- induced pluripotent stem cells (iPSCs)- normal specialised cells that have been genetically reprogrammed to become undifferentiated.
- RNA polymerase must bind to DNA molecule upstream from promoter
- Will only bind weakly, needs DNA-binding proteins called transcription factors
- Transcription factors binds to specific binding site that binds to a particular DNA sequence of the promoter.
Steriod hormones control protien synthesis they do this by transcription factors
- oestrogen crosses membrane by lipid diffusion
- it binds to receptor protein, forming receptor-hormone complex- TF
- active 'TF' diffuses into nucleus through nulcear pores
- binds to specific base on DNA promoter
- stimulate RNA polymerase to transcribe the gene
CONTROL of mRNA by RNA INTERFERENCE
- The quicker the mRNA is broken down, the less protein is made
- control is carried by small interfering RNA (siRNA) and process is called RNA interference (RNAi)
- siRNA short double stran RNA molecule which is made by special regulatory genes. made by a single strand RNA, which then folds by complementary base pairing.
- siRNA binds to protein called RNA-induced silencing complex (RISC)
- RISC breaks down the double stranded siRNA,so a single strand remains attached to RISC but other is discarded.
- RISC-RNA complex now binds to the mRNA in the cytoplasm by complementary base pairing
- Binding causes RISC to cut mRNA into two
- mRNA no longer used so it is broken down by nuclease enzymes.
RESTRICTION ENZYMES & DNA LIGASE
Enzymes that cut the DNA at a specific site
- cut ends are 'sticky' because they have short stretch of single stranded DNA with complementary sequence
- sticky ends will stick (anneal) byt complementary base pairing- hydrogen bonding
- will only stick if the same resitriction enzymes are used
- restriction enzymes are highly specific
- naturally produce by viruses
- enzymes repair broken DNA by joining two nucleotides in the DNA strand
- commonly used in genetic modification
- they produce phosphodiester bonds
- synthesis DNA from RNA template
- biotechnology uses it to make artificial genes called complementary DNA (cDNA)
- cDNA is much shorter than original DNA
Uses in biotechnology
- makes genes without introns
- makes stable copy of a gene since DNA is readily broken down by enzymes
- makes genes easier to find.
POLYMERASE CHAIN REACTION (PCR)
Used to copy (amplify) DNA sample as small as a sing molecule
- heat the sample to 95c for two minutes to break hydrogen bonds between base pairs and seperate the two strands
- Add primer to the mixture and allow to cool. Primer are short lenght of single stranded DNA that anneal by complementary sequences
- Enzyme DNA polymersae require double strands to start transcription
- it ensures that only a specific target sequence is copied
- DNA polymerase can now build new strands. Enzymes used in PCR grow on hot springs, advantage is that it will not denature when using high temperature
- Any contaminated DNA will also be amplified.
ELECTROPHORESIS & DNA SQUENCING
- form of chromotography used to seperate differenct piece of DNA bases on lenght
- each nucleotide contains negatively charged phosphate group
- shorter the fragement the further it travels
- in each test tube add sample, radioactive primer, the four nucleotides and DNA prolymerase
- modified dideoxy nucleotides that cannot form phosphodiester bonds so stop further synthesis of DNA
- DNA polymerase synthesis many compy of DNA. The dna will range in lenght - in test tube A all fragments will stop at an A nucleotide
- contents are now run down side by side on electrophoresis gel.
- diagram of a piece of DNA marked with the location of site where it is cut by restriction enzymes
- usually piece of DNA is cut with two different restriction enzymes. both on there own and together
- run on electrophoresis
- first lane - 'DNA ladder' mixture of DNA fragments of known size
- lenght of each fragments can be measured
- spaces between fragments is the number of cuts which have been made
- DNA probe,simple short lenght od single-stranded DNA with a label attached
- will anneal to complementary sequence- hybrid DNA
- process called hybridisation
- PCR is used to amplify DNA, restriction enzyme is present and electrophoresis is done
- Gel placed in alkali solution which breaks hydrogen bonds causing the two strands to seperate
- nylon sheet is placed on top of gell, alkali solution is drawn up through gel to paper towel by capillary action, bringing the DNA with it. negatively-chraged DNA sticks to positively-charges nylon membrane
- nylon treated with UV light to fix DNA molecules
- placed in a bag, probes anneal to DNA. which forms a hybrid DNA molecule
- Difference found in non-coding DNA
- non-coding DNA is mainly made up of long repetitive sequence
- Amplified DNA of VNTR region is digested by restriction enzymes. Same recognition sequence should be present in different sample but lenght will depend on how many repeats
- Run down on an electrophoresis gel to seperate fragment
- visualised by southern blot
- In genetic engineering a vector is a lenght of DNA that carries the gene we want into a host cell
- Plasmid are commonly used vectors
- They are small so easy yo handle in test tube
- Restriction enzymes alcut the gene from donor DNA
- Same restriction enzyme used to cut in middle of one marker gene
- Will anneal due to complementary sticky ends
- DNA ligase will also be used
- Hybrid DNA is produced
Maker genes are used to find which cells have actually taken up the hybrid vector.
- First maker genes distinguishes between cells that have taken up the plasmid from those who havent
- Second will distinguish between cell that have taken up the hybrid and original plasmid.
- test is done on replica plate
- Colonies that grow on the first (tetracyljne) plate but not on the replica (ampicillin) plate are the ones we want as it has hybrid DNA.