genetics

RNA SYNTHESIS

• 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.

• 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

PROTEIN SYNTHESIS

• 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
  • viruses.

• 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.

Stem cells

• 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

• 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.

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

DNA LIGASE

• 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.

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

• 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

DNA sequencing

• 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.