MODERN DNA SEQUENCING

What & Why? • “Sequencing” means finding the order of nucleotides on a piece of DNA . • Nucleotide order determines Amino acid order, and by extension, protein structure and function (proteomics) • An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or animal

What & Why, Ctd. • Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment • DNA technology is also extended to environmental, agricultural and forensic applications

DNA Sequence variation can change the Protein produced by a particular gene Gene A from per son 1

Codon resul t s in part icular Am ino Acid (AA) sequ ence

GCA Ala

AGA Arg

GAT Asp

AAT Asn

TGT Cys

Gene A from per son 2

Codon chan ge m akes no differen ce in AA (redu ndant code) Codon chan ge result s in different AA sequ ence

GCG

AGA

GAT

AAT

TGT

Ala

Arg

Asp

Asn

Cys

GCA Ala

AAA Lys

GAT Asp

AAT Asn

TGT Cys

Gene A from per son 3

Simple point mutations such as this can cause altered protein shape and function. Diseases such as Sickle Cell Anaemia and Cystic Fibrosis are caused by point mutations

Historically there are two main methods of DNA sequencing: Maxam & Gilbert, using chemical sequencing Sanger, using dideoxynucleotides. Modern sequencing equipment uses the principles of the Sanger technique.

The Sanger Technique • Uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc) • These are molecules that resemble normal nucleotides but lack the normal -OH group.

• Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops. Normally, this would be where another phosphate Is attached, but with no OH group, a bond can not form and replication stops

The Sanger method requires • Multiple copies of single stranded template DNA • A suitable primer (a small piece of DNA that can pair with the template DNA to act as a starting point for replication) • DNA polymerase (an enzyme that copies DNA, adding new nucleotides to the 3’ end of the template • A ‘pool’ of normal nucleotides • A small proportion of dideoxynucleotides labeled in some way ( radioactively or with fluorescent dyes)

• The template DNA pieces are replicated, incorporating normal nucleotides, but occasionally and at random dideoxy (DD) nucleotides are taken up. • This stops replication on that piece of DNA • The result is a mix of DNA lengths, each ending with a particular labeled DDnucleotide. • Because the different lengths ‘travel’ at different rates during electrophoresis, their order can be determined.

Terminatio n during Replication DNA G SEQUENCE 3’ PRIMER C 5’ NO OF 1 BASES

G terminated A terminated

C

A

T

G

T

A

2

3

4

T

G

G

G

A

A

C

C

5

6

7

8

9

10

11

12

CG T A A CCT T G CG T A A CCT T G G CG T A A

Tterminated

CG T A A CC T CG T A A CC T T

C terminated

CG T A A C CG T A A C C CG T A A C C C

• Originally four separate sets of DNA, primer and a single different DD nucleotide were produced and run on a gel. • Modern technology allows all the DNA, primers, etc to be mixed and the fluorescent labeled DDnucleotide ‘ends’ of different lengths can be ‘read’ by a laser. • Additionally, the gel slab has been replaced by polymer filled capillary tubes in modern equipment • This is the basis of the sequencer used at the Centre for Genomics and Proteomics in the School of Biological Sciences at the University of Auckland, as seen in the next slides.

Step 1- Before submission for sequencing DNA purity & concentration is checked with the ‘Nanodrop’

A Nanodrop readout of known concentration to be run as a control

Step 2 -Samples are received and stored in the refrigerator and a request filed

Cost? • Cost is dependant on a number of factors but typically in 2003: • Each tube of sample DNA costs $27 to run. • An entire set of 96 tubes from one source (the capacity of the present equipment) costs $960. • The methods used will readily analyze DNA fragments of 500-1000 bases in length, depending on the quality of DNA used • Note – the dye alone to run 5000 reactions costs $61,000

Samples arrive in Eppendorf tubes

Step 3 - paperwork. Each request is assigned a ‘well’ in the sample tray and volumes of primers, water, dye, etc are calculated. A typical ‘run’ has samples from a number of researchers

Step 4- Samples are agitated then centrifuged in an Ultracentrifuge to be sure they are in the bottom of their Eppendorf tubes.

Step 5 - Reagents, etc • Each reaction requires several reagents: • Specific primers for the DNA in question • Fluorescent Dye attached to DD nucleotides (Big Dye) • Deionised water • DNA polymerase • Additionally, a ‘control’ sample of a known DNA is prepared so it can run at the same time as the experimental DNA

Micropipettors come in a range of sizes. They have disposable tips that hold tiny amounts of required reagents.

Step 6 - Preparing the wells • The Sample wells are loaded with DNA to be sequenced. Great care needs to be taken to ensure that each sample goes into its assigned well. • Reagents are added (water, dye, primers) in required amounts • The sample wells are ‘spun’ to ensure that the DNA and reagents are mixed and at the bottom of the sample wells.

Sample tray and micropipettor. Each tray holds 96 samples

Step 7 - The samples are run through a cycle sequencing process to get the fluorescent dyes incorporated by the DNA. The DNA and reagents are alternately heated and cooled over a 2 1/2 hour period.

Step 8 - Sample purification to get rid of extra dye and salts • Unincorporated dye and salts can interfere with DNA analysis and need to be removed • Samples are centrifuged, precipitated with 95% ethanol, centrifuged again, and drained • The process is repeated with 70% ethanol • Dry samples are either analyzed immediately or stored in the dark (light degrades the fluorescent dyes used) • Just before sequencing formamide is added to ensure that the DNA remains linear

Entering data from the record sheet into the Sequencer software programme

Step 10- The sequencer is warmed up, reagents are refreshed and the sample tray is inserted

Capillary tubes Reagents

Sample tray goes here

Inside the sequencer

The Sequencer Apparatus • Each sample tray has 96 wells (1 per sample), and the analyzer (3100 model) has the capacity to analyze 16 wells at a time • Robotic apparatus moves the sample tray so each of the 16 wells is in contact with a separate capillary tube filled with a polymer this replaces a lane on an electrophoresis gel • Labeled DNA from that well moves up the capillary tube, with smaller labeled fragments moving more quickly than longer ones

The Sequencer, II • A laser ‘reads’ the fluorescent label on each fragment as it passes up the capillary tube • It takes 4 hours to ‘run’ 16 samples. The robotics then move the capillaries through a cleaning phase and move the tray of samples so the next 16 samples are processed. It takes 24 hours to process 96 samples. • Electronic signals from the laser go to a sequencer programme and are converted into an electronic file of the code

The Sequencer, III • Ambiguous readings are indicated by an ‘N’ • Printouts of each record are checked for a ‘pass’ or ‘fail’ (failure may be due to degraded primer, insufficient DNA, etc) • Records are stored in a computer ‘drop box’ for electronic collection by university staff, or are mailed to off-campus customers

A schematic of sequencing

A Sequence print-out from a control sample

What Next? • In some instances the section of DNA analyzed may be all that is needed for some research project • In other cases the DNA fragment’s code is matched to overlapping sections of other fragments. This eventually can result in the entire genome of an organism. • Matching is done by the use of sophisticated software • The National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) maintains a number of searchable public DNA databases • Sequencing is starting to be done on ‘gene chips’, microarrays of known DNA segments- this area of study is evolving rapidly

Acknowledgements Thanks go to: Kristine Boxen of the Centre for Genomics & Proteomics Craig Millar, SBS, Auckland University

References • Campbell, N., Reece, J.B., 2002, Biology 6th ed., Benjamin Cummings, San Francisco • Drlica, K., 1997, Understanding DNA & Gene Cloning, 3rd ed., John Wiley & Sons, NY • Kreuzer, H., Massey, A., 2001, Recombinant DNA & Biotechnology, 2nd ed., ASM Press, Washington, DC • Turner, P.C.,et.al., 1997, Instant Notes in Molecular Biology, Bios, Oxford • www.ncbi.nlm.nih.gov/about/primer/genetics_molecular.html (slide 32), used by kind permission • Photographs by L Macdonald, 2003

The End- Or Just the Beginning?

Compiled by Linda Macdonald For NCEA Biology A.S. 3.6 With support from the Royal Society Science, Mathematics &Technology Teacher Fellowship Scheme