Presentation on theme: "DNA and Gene Cloning DNA revision Gene cloning"— Presentation transcript:
1 DNA and Gene Cloning DNA revision Gene cloning cDNA and reverse transcriptaseRestriction endonucleases
2 Collect some nucleotides The 5’ prime and 3’ prime ends of the bases must be the right way round!
3 Collect the following: 3 yellow bases with a phosphate and a black sugar3 yellow bases with a phosphate and a red sugar3 green bases with a phosphate and a black sugar3 green bases with a phosphate and a red sugar4 blue bases with a phosphate and a black sugar4 blue bases with a phosphate and a red sugar4 orange bases with a phosphate and a black sugar4 orange bases with a phosphate and a red sugar
4 Build: Blue Orange Yellow Green With nucleotides containing black sugars:BlueOrangeYellowGreen
5 Join some nucleotides together to form some DNA. Green (Guanine) pairs with yellow (Cytosine)Blue (Adenine) pairs with orange (Thymine)
6 In your group and using the model… Describe how DNA replicatesKey terms:- DNA helicase- DNA polymerase- Complementary basesDescribe how DNA is used as a template for protein productionKey terms:DNA helicaseRNA polymeraseComplementary basesTranscriptionTranslation
8 DNA Technology Isolation – of the DNA containing the required gene Insertion – of the DNA into a vectorTransformation – Transfer of DNA into a suitable hostIdentification – finding those host organisms containing the vector and DNA (by use of gene markers)Growth/Cloning – of the successful host cells
9 DNA Technology Isolation – of the DNA containing the required gene Insertion – of the DNA into a vectorTransformation – Transfer of DNA into a suitable hostIdentification – finding those host organisms containing the vector and DNA (by use of gene markers)Growth/Cloning – of the successful host cells
10 Learning Objectives: Stage 1 – Producing DNA fragments How is complementary DNA made using reverse transcriptase?How are restriction endonucleases used to cut DNA into fragments?
11 Reverse Transcriptase A group of viruses called retroviruses (e.g. HIV) contain an enzyme called reverse transcriptase.It is used to turn viral RNA into DNA so that it can be transcribed by the host cell into proteins.
12 Producing DNA copies from mRNA Boardworks A2 BiologyGenetic TechnologiesProducing DNA copies from mRNATeacher notesSee the Boardworks AS Biology ‘Infectious Diseases’ presentation for more information about how HIV uses reverse transcriptase to replicate its viral components in a host’s cell.
13 Reverse Transcriptase DNA polymeraseReverse transcriptase makes DNA from an RNA template – it does the opposite of transcription.
14 Using reverse transcriptase β-cells from Islets of Langerhans in the human pancreas.Extract mature mRNA coding for insulin.A single stranded complementary DNA strand (cDNA) isformed using reverse transcriptase and the mRNA template.Single stranded cDNA is used to form double strandedDNA using DNA polymerase.This forms a double stranded copy of the human insulingene.
15 Restriction EnzymesBacteria contain restriction enzymes in order to protect themselves from invading viruses.Restriction enzymes are used by bacteria to cut up the viral DNA.These enzymes cut DNA at specific sites – this property can be useful in gene technology.
16 Restriction Endonucleases Have highly specific active sites.Cut DNA at specific sites – about 4-8 base pairs long – these are called recognition sites.Recognition sites are palindromic - the sequence and its complement are the same but reversed.E.g. -GAATTC--CTTAAG-
17 Assemble the following sequence: With any colour of sugar:5’ – GTCGACCCGGGTCGACA – 3’Then complete the complementary strand. Don’t forget to orient it correctly!G = green C = yellow A = blue T = orange
18 Restriction enzymes Sma1 cuts between the C and the G of CCCGGG. Taq1 cuts between the T and the C of TCGA.What do you notice when you perform each of these cuts?What if you use both enzymes?
19 Producing DNA copies by cutting DNA Boardworks A2 BiologyGenetic TechnologiesProducing DNA copies by cutting DNATeacher notesRestriction endonucleases produced in bacteria are thought to have evolved as a defensive mechanism, to protect against invading viruses. The enzyme could cut the viruses’ genetic material into fragments preventing it from replicating. The host’s DNA is methylated by another enzyme in order to protect it from the activities of the restriction endonuclease.Certain sequences of DNA are the same whether they are read 5' to 3' or 3' to 5'. These sequences are called palindromic.
20 Restriction Enzymes – “Blunt Ends” Some restriction enzymes cut straight across both chains forming blunt ends.
21 Restriction Enzymes – “Sticky Ends” Most restriction enzymes make a staggered cut in the two chains, forming sticky ends.
22 Sticky Ends…Sticky ends have a strand of single stranded DNA which are complementary to each other.They will join with another sticky end but only if it has been cut with the same restriction enzyme.
23 How do we know which section of DNA to use? We use a process called Southern blotting.Involves gel electrophoresis, binding of DNA to a membrane, and visualisation of the DNA section of interest using a DNA probe.Animation
24 Gel ElectrophoresisA sample containing DNA fragments of different sizes is placed at one end of an agarose gel.A current is passed through the gel, causing negatively-charged DNA (due to the phosphate groups) to be attracted to the positive electrode.Smaller fragments move faster and further than larger fragments.
25 Southern BlottingThe gel is treated with a basic solution to separate the DNA strands (breaks the H-bonds).A nylon membrane is placed over the gel and many paper towels are placed on top.Over many hours, the solution is drawn up into the paper towels, bringing the DNA with it. The DNA sticks to the membrane.
26 DNA HybridisationA radioactively-labelled DNA probe (single stranded DNA with a sequence complementary to the section of interest) is washed over the membrane.The probe binds with any fragments of DNA containing the complementary base sequence.X-ray film is used to detect the presence of hybridised DNA fragments.
27 Restriction mappingAfter gel electrophoresis has been performed, we may wish to work out in what order different fragments appear in the DNA sample.A restriction map shows us where the fragments are located.
28 Boardworks A2 Biology Studying Genomes Locating genesTeacher notesMap I is correct because the electrophoresis shows lines at 0.4, 0.8 and 5.8 kb as predicted in the previous stage of the animation.28
29 Locating a specific gene Boardworks A2 BiologyStudying GenomesLocating a specific geneOne approach to locating genes is to use a DNA probe.This is a short section of DNA that has been labelled, for example with radioactive phosphorous or a fluorescent marker.The DNA is of a known sequence corresponding to the gene being looked for, for example the cystic fibrosis gene during clinical screening.fluorescent markerDNA probetarget gene29
30 Boardworks A2 Biology Studying Genomes Using a gene probeBefore using a gene probe the DNA needs to be heated to separate the two strands.The temperature is then reduced so that the probe can ‘anneal’ or hybridize with the sample DNA as a result of complementary base pairing.hybridized probeThe location of the gene is then identified. The method used depends on the method of labelling. Radioactive tags are located by exposing the DNA to a photographic plate; fluorescent tags are located by using UV light in a fluorescent microscope.
31 Carry out the ‘Restriction mapping and Southern blotting’ activity.