genetics

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  • Created by: ricka
  • Created on: 21-04-13 14:25

TRANSCRIPTION

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.
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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
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TRANSLATIONS

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
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GENE MUTATION

  • 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.
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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.

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. 
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TRANSCRIPTION FACTOR

  • 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
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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.
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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

DNA LIGASE

  • enzymes repair broken DNA by joining two nucleotides in the DNA strand
  • commonly used in genetic modification
  • they produce phosphodiester bonds
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REVERSE TRANSCRIPTASE

  • 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.
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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.
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ELECTROPHORESIS & DNA SQUENCING

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.
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RESTRICTION MAPPING

  • 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
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SOUTHERN BLOT

  • 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 
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GENETIC FINGERPRINTING

  • 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
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VECTORS

  • 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
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MAKER GENES

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.
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