Presentation on theme: "DNA sequencing by the Sanger method"— Presentation transcript:
1 DNA sequencing by the Sanger method The standard DNA sequencing technique is the Sanger method,named for its developer, Frederick Sanger, who shared the 1980Nobel Prize in Chemistry. This method begins with the use ofspecial enzymes to synthesize fragments of DNA that terminatewhen a selected base appears in the stretch of DNA beingsequenced. These fragments are then sorted according to sizeby placing them in a slab of polymeric gel and applying anelectric field -- a technique called electrophoresis. Because ofDNA's negative charge, the fragments move across the gel towardthe positive electrode. The shorter the fragment, the faster itmoves. Typically, each of the terminating bases within thecollection of fragments is tagged with a radioactive probe foridentification.
2 DNA sequencing example Problem Statement: Consider the following DNAsequence (from firefly luciferase). Draw the sequencinggel pattern that forms as a result of sequencing thefollowing template DNA with ddNTP as the capper.atgaccatgattacg...Solution:Given DNA template: '-atgaccatgattacg...-3'DNA synthesized: '-tactggtactaatgc...-5'
3 DNA sequencing example Given DNA template: 5'-atgaccatgattacg...-3'DNA synthesized: '-tactggtactaatgc...-5'Gel pattern:lane ddATP | W | | || |lane ddTTP | W | | | | | |lane ddCTP | W | | | |lane ddGTP | W || | |Electric FieldDecreasing sizewhere "W" indicates the well position, and "|"denotes the DNA bands on the sequencing gel.
4 A sequencing gelThis picture is a radiograph. The dark color of the lines isproportional to the radioactivity from 32P labeled adenonsinein the transcribed DNA sample.
5 Reading a sequencing gel You begin at the right, which are the smallest DNA fragments.The sequence that you read will be in the 5'-3' direction.This sequence will be exactly the same as the RNA thatwould be generated to encode a protein. The difference is thatthe T bases in DNA will be replaced by U residues. As an example,in the problem given, the smallest DNA fragment on the sequencinggel is in the C lane, so the first base is a C. The next largest bandis in the G lane, so the DNA fragment of length 2 ends in G.Therefore the sequence of the first two bases is CG.The sequence of the first 30 or so bases of the DNA are:CGTAATCATGGTCATATGAAGCTGGGCCGGGCCGTGC....When this is made as RNA, its sequence would be:CGUAAUCATGGUCAUAUGAAGCUGGGCCGGGCCGUGC....Note that the information content is the same, only the T's havebeen replaced by U's!.
7 Translating the DNA sequence The order of amino acids in any protein is specificed by theorder of nucleotide bases in the DNA.Each amino acid is coded by the particular sequence of three bases.To convert a DNA sequenceFirst, find the starting codon. The starting codon is alwaysthe codon for the amino acid methionine. This codon isAUG in the RNA (or ATG in the DNA):GCGCGGGUCCGGGCAUGAAGCUGGGCCGGGCCGUGC....MetIn this particular example the next codon is AAG. The first base(5'end) is A, so that selects the 3rd major row of the table. Thesecond base (middle base) is A, so that selects the 3rd column ofthe table. The last base of the codon is G, selecting the last line inthe block of four.
8 Translating the DNA sequence This entry AAG in the table is Lysine (Lys).Therefore the second amino acid is Lysine.The first few residues, and their DNA sequence, are as follows(color coded to indicate the correct location in thecodon table):Met Lys Leu Gly Arg …AUG AAG CUG GGC CGG GCC GUG C..This procedure is exactly what cells do when they synthesizeproteins based on the mRNA sequence. The process of translationin cells occurs in a large complex called the ribosome.
9 Automated procedure for DNA sequencing A computer read-out of the gel generates a “false color” imagewhere each color corresponds to a base. Then the intensities aretranslated into peaks that represent the sequence.
10 High-throughput seqeuncing: Capillary electrophoresis The human genome projecthas spurred an effort todevelop faster, higherthroughput, and lessexpensive technologiesfor DNA sequencing.Capillary electrophoresis(CE) separation has manyadvantages over slab gelseparations. CE separations are faster and are capable of producinggreater resolution. CE instruments can use tens and evenhundreds of capillaries simultaneously. The figure show a simpleCE setup where the fluorescently-labeled DNA is detected as itexits the capillary.Sheath flowLaserFocusinglensSheath flow cuvetteBeam blockPMTCollection LenscCollection Lenscfilter
11 Sieving matrix for CEIt is not easy to analyze DNA in capillaries filled only withbuffer. That is because DNA fragments of different lengthshave the same charge to mass ratio. To separate DNA fragmentsof different sizes the capillary needs to be filled with sievingmatrix, such as linear polyacrylamide (acrylamide polymerizedwithout bis-acrylamide).This material is not rigid like a cross-linked gel but looks much like glycerol. With a little bit ofeffort it can be pumped in and out of the capillaries. To simulatethe separation characteristics of an agarose gel one can usehydroxyethylcellulose. It is not much more viscous then waterand can easily be pumped into the capilliaries.