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Section 4 Lesson 5 – Human Therapeutics

Section 4 Lesson 5 – Human Therapeutics. PCR Quiz. What is special about Taq ?. It is a thermal stable enzyme able to operate at higher temperatures. What temperature is best for the synthesis stage in the process?. 72˚C. 3. Give 2 possible applications of this technique.

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Section 4 Lesson 5 – Human Therapeutics

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  1. Section 4Lesson 5– Human Therapeutics

  2. PCR Quiz What is special about Taq? It is a thermal stable enzyme able to operate at higher temperatures. What temperature is best for the synthesis stage in the process? 72˚C 3. Give 2 possible applications of this technique. DNA sequencing, diagnosis of hereditary illnesses, diagnosis of infectious diseases, phylogeny, genetic fingerprinting 4. What do primers do? Anneal to target sequences 5. Which way does Taq transcribe (3’ – 5’ OR 5’ – 3’)? 3’ – 5’

  3. Dideoxy chain-termination Method This is sometimes know as the Sanger method as it was first developed in 1977 by Frederick Sanger in Cambridge. Sanger sequencing is modelled after the natural process of DNA replication, and it uses ‘dummy’ nucleotides to stop replication whenever a specific nucleotide is encountered. Because this truncated replication occurs over and over again, nucleic acids of varying lengths accumulate and can be used to determine the position of each nucleotide in the sequence.

  4. How Does it Work? Sanger Method Video Sanger Sequencing

  5. How Does it Work? PCR is used to amplify the section of DNA that is to be sequenced. The DNA is then heated to denature it and a primer is added that corresponds to the start of the sequence. DNA polymerase dNTPs are also added. These are deoxynucleotide triphosphates, or dNTPs (where the "N" is a placeholder for A, T, G, or C). These will create a normal copy of the strand. Crucially at this stage dideoxynucleotidetriphosphates, or ddNTPsare also added. These are chemically slightly different (lacking an –OH group). This difference prevents them from binding to the next dNTP (or ddNTP) and caused transcription to STOP. These were radioactively labelled so they could be spotted on the gel at the end. Originally 4 flasks were set up – each with a different ddNTP added. Now this is done in a single flask. The result is a mixture of strands of varying lengths each ending in a terminal ddNTP that indicates whether it is A,T,G or C. Gel electrophoresis is used to separate out the different lengths of DNA.

  6. deoxyribonucleoside triphosphate dideoxyribonucleoside triphosphate Prevents strand extension at 3’ end Allows strand extension at 3’ end Incorporation of this nucleotide results in chain termination.

  7. Separating DNA Fragments DNA fragments can be separated by gel electrophoresis Largest fragments Smallest fragments DNA moves to the positive terminal due to it’s overall negative charge + gel with DNA fragments

  8. Gel Electrophoresis

  9. Reading a Gel Use the handout of the gel to read the sequence of this DNA fragment.

  10. Automated Method The original method worked well but was time consuming. It was later adapted to use fluorescent labelling instead of radioactive labelling. This method allowed the labels to be detected as they ran out the end of the gel in the correct order.

  11. Next Generation Sequencing Wiki - next generation info table

  12. Comparative Genome Analysis As our knowledge and understanding of individual genomes has increased, our ability to make comparisons between genomes has also increased. The monograph tells us that in general, the more complex the organism is the more complex the genes are and the more genes are present. More recent research has proven this to not always be the case but that further comparative analysis is needed.

  13. Your Tasks Add the red terms to your glossary. Past Paper Questions due Monday March 4th. 2005 MC Q11,12 2006 MC Q 12 2007 Q7 2008 MC Q 10,11,12 2009 MC Q 12, ER Q8B 2010 MC Q 11,12,13

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