DNA sequencing methods Content History of DNA sequencing

DNA sequencing methods

Content History of DNA sequencing Some methods Dideoxy sequencing method How to visualize DNA fragments? Analysis of sequencing products Microarrays

History of DNA sequencing

History of DNA sequencing

Methods of sequencing Sanger dideoxy (primer extension/chain-termination) method: most popular protocol for sequencing, very adaptable, scalable to large sequencing projects Maxam-Gilbert chemical cleavage method: DNA is labelled and then chemically cleaved in a sequence-dependent manner. This method is not easily scaled and is rather tedious Pyrosequencing: measuring chain extension by pyrophosphate monitoring

For dideoxy sequencing you need: Single stranded DNA template A primer for DNA synthesis DNA polymerase Deoxynucleoside triphosphates and dideoxynucleotide triphosphates

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

Sanger dideoxy sequencing: basic method b) Extend the primer with DNA polymerase in the presence of all four dNTPs, with a limited amount of a dideoxy NTP (ddNTP) ’ 3’ Direction of DNA polymerase travel DNA polymerase incorporates ddNTP in a template-dependent manner, but it works best if the DNA pol lacks 3’ to 5’ exonuclease (proofreading) activity

Sanger dideoxy sequencing: basic method 5’ 3’ 5’ 3’ T T T T ddA ddA ddA ddA ddATP in the reaction: anywhere there’s a T in the template strand, occasionally a ddA will be added to the growing strand

How to visualize DNA fragments? Radioactivity Radiolabeled primers (kinase with 32P) Radiolabelled dNTPs (gamma 35S or 32P) Fluorescence ddNTPs chemically synthesized to contain fluors Each ddNTP fluoresces at a different wavelength allowing identification

Analysis of sequencing products: Polyacrylamide gel electrophoresis--good resolution of fragments differing by a single dNTP Slab gels: as previously described Capillary gels: require only a tiny amount of sample to be loaded, run much faster than slab gels, best for high throughput sequencing

DNA sequencing gels: old school Analyze sequencing products by gel electrophoresis, autoradiography Different ddNTP used in separate reactions Radioactively labelled primer or dNTP in sequencing reaction

cycle sequencing: denaturation occurs during temperature cycles 94°C:DNA denatures 45°C: primer anneals 60-72°C: thermostable DNA pol extends primer Repeat 25-35 times Advantages: don’t need a lot of template DNA Disadvantages: DNA pol may incorporate ddNTPs poorly

An automated sequencer The output

Current trends in sequencing: It is rare for labs to do their own sequencing: --costly, perishable reagents --time consuming --success rate varies Instead most labs send out for sequencing: --You prepare the DNA (usually plasmid, M13, or PCR product), supply the primer, company or university sequencing center does the rest --The sequence is recorded by an automated sequencer as an “electropherogram”

~160 kbp ~1 kbp Assemble sequences by matching overlaps BAC sequence BAC overlaps give genome sequence BREAK UP THE GENOME, PUT IT BACK TOGETHER

Bioinformatics: making sense of biological sequence New DNA sequences are analyzed for ORFs (Open Reading Frames: protein) Any DNA or protein sequence can then be compared to all other sequences in databases, and similar sequences identified There is much more -- a great diversity of programs and databases are available

Massively parallel measurements of gene expression: microarrays Defining the “transcriptome” The northern blot revisited Detecting expression of many genes: arrays A typical array experiment What to do with all this data? Brown and Botstein (1999) “Exploring the new world of the genome with DNA microarrays” Nature Genetics 21, p. 33-37.

DNA RNA protein genome “transcriptome” “proteome” (we have this) (we want these)

The value of DNA microarrays for studying gene expression Study all transcripts at same time Transcript abundance usually correlates with level of gene expression--much gene control is at level of transcription Changes in transcription patterns often occur as a response to changing environment--this can be detected with a microarray

A yeast array experiment vegetative sporulating Isolate mRNA Prepare fluorescently labeled cDNA with two different-colored fluors hybridize read-out

Example microarray data Green: mRNA more abundant in vegetative cells Red: mRNA more abundant in sporulating cells Yellow: equivalent mRNA abundance in vegetative and sporulating cells

What to do with all that data? Overarching patterns may become apparent Organize data by hierarchical clustering, profiling to find patterns Display data graphically to allow assimilation/comprehension