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DNA Sequencing

DNA Sequencing. Fred Sanger. The elegant idea behind DNA sequencing . In the 1970’s, Sanger’s group discovered a fundamentally new method of 'reading' the linear DNA sequence using special bases called chain terminators or dideoxynucleotides .

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DNA Sequencing

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  1. DNA Sequencing

  2. Fred Sanger The elegant idea behind DNA sequencing In the 1970’s, Sanger’s group discovered a fundamentally new method of 'reading' the linear DNA sequence using special bases called chain terminators or dideoxynucleotides. This method is still in use today it is called:Sanger dideoxynucleotides chain-termination method. What is the basis of Sanger’s method? Shared with Walter Gilbert and Paul Berg

  3. Dideoxynucleotidessequencing requires: Single stranded DNA template A primer for DNA synthesis DNA polymerase Deoxynucleosidetriphosphates and dideoxynucleotidetriphosphates

  4. DNA polymerase synthesizes a second DNA strand from a labeled (radioactive) primer. • DNA polymerase always adds new bases to the 3’ end of a primer that is base-paired to the template DNA. • DNA polymerase is modified to eliminate its editing function • chain terminator nucleotides: dideoxy nucleotides (ddNTPs), which lack the -OH group on the 3' carbon of the deoxyribose. When DNA polymerase inserts one of these ddNTPs into the growing DNA chain, the chain terminates, as nothing can be added to its 3' end.

  5. Sanger dideoxy sequencing--basic method Single stranded DNA 5’ 3’ ★5’ 3’ a) Anneal the primer

  6. Dideoxy Nucleotide

  7. Sanger dideoxy sequencing: basic method 5’ 3’ T T T T 3’ 5’ ddATP in the reaction: anywhere there’s a T in the template strand, occasionally a ddA will be added to the growing strand ddA ddA ddA ddA sequencing products separated by gel electrophoresis and detected by autoradiography

  8. How to visualize DNA fragments? • Radioactivity • Radiolabeled primers (with 32P) • Fluorescence • ddNTPs chemically synthesized to contain fluors • Each ddNTP fluoresces at a different wavelength allowing identification

  9. DNA sequencing gels: Different ddNTP used in separate reactions • Sequencing is done by having 4 separate reactions, one for each DNA base. • All 4 reactions contain the 4 normal dNTPs, but each reaction also contains one of the ddNTPs. • In each reaction, DNA polymerase starts creating the second strand beginning at the primer. • When DNA polymerase reaches a base for which some ddNTP is present, the chain will either: • - terminate if a ddNTP is added, or • - continue if the corresponding dNTP is added.. Radioactively labeled primer or dNTP in sequencing reaction Analyze sequencing products by gel electrophoresis and autoradiography

  10. 5’ 3’

  11. 5’ 3’ DNA read

  12. http://www.ncbi.nlm.nih.gov

  13. 1 2 3 4

  14. Enter query sequence, select parameters, click blast

  15. EXERCISE #1 Read the autoradiographs from bottom up. Start at the arrow and read up about 20 nucleotides. What are the names of these genes? What species do these genes belong to? EXERCISE #2 Read sample2 approximately 6 cm from bottom of the strip. 5’---GGACGACGGTATGGAATAGAGAGGAAGTTCCTC---3’ What is the name of this gene? What species do this gene belong to? What strand have you read?

  16. EXERCISE #3 Read the sequence from sample 3. What is the name of this gene? How many amino acids does this gene have? More information can be accessed by clicking on Genbank accession number. EXERCISE #4 A- Read sample 4 from bottom of the strip. Record the DNA sequence. B- Then move a third of the way up and read a portion of this sequence What are the corresponding names of these 2 genes? What are the functions of the 2 proteins encoded by these 2 genes? How do these 2 proteins interact in a living cell?

  17. http://www.youtube.com/watch?v=SRWvn1mUNMA http://www.dnalc.org/resources/animations/cycseq.html

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