Lecture 8: Molecular Markers and mapping Next generation DNA sequencing DNA polymorphism: the basis of molecular markers

Methods of detection and application (diagnosis, finger-printing)

Read 391-408 Fig. 11.3; 11.4; 11.6-8; 11.10-15 Table 11.1
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Personal Genome Project (PGP) Goal: sequencing full genome of individuals at $1000
Human Genome Project (HGP) (total 3 billion $) : Motivated 100X reduction in cost (10 $ per base to 10 base per 1$.) Personal Genome Project(PGP: has motivated the development of Ultra-Low-Cost-Sequencing (ULCS).

ULCS under development

Microelectrophoretic sequencing Hybridization sequencing Cycle-array sequencing on amplified molecules (SBS) Pyrosequencing (454) Reversible terminator sequencing (Solexa) Restriction digestion and ligation (MPSS-Lynx) Fluorescent In Situ Sequencing (FISSEQ) (Multiplex in space and time; Avoidance of bacterial clones) Nanopore sequencing

Detects extension through luciferase-based real time monitoring of pyrophosphate release.

Pyrosequencing

Pyrosequencing invented by Mostafa Ronaghi, furthered by Biotage

Step 1: PCR amplified ssDNA templates is hybridized to a sequencing primer and incubated with the enzymes: DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5 phosphosulfate (APS) and luciferin. Step 2: The addition of one of the four deoxynucleotide triphosphates (dNTPs) initiates the second step. DNA polymerase will catalyze the incorporation of dNTP onto the template if it is complementary. It is important to note that if there is an incorporation there will be a release of pyrophosphate (PPi) equivalent to the amount of dNTP incorporated. Step 3: ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5 phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and this can be analyzed in a program. Each light signal is proportional to the number of nucleotides incorporated. Step 4: To continue the sequencing the degrading of nucleotides is essential. Apyrase is a nucleotide degrading enzyme and does in this step clean the solution from all dNTP. Step 5: New nucleotides can be added and a new cycle can start. Ronaghi, M., Uhlen, M., and Nyren, P. 1998. Science 281: 363. A sequencing method based on real-time pyrophosphate.

Megaclone technology (invented by S. Brenner and patented by Lynx Therapeutics)

http://mpss.udel.edu/tutorial/flash/megaclone.swf
Megaclone transforms a sample containing millions of DNA molecules into one made up of millions of micro-beads, each of which carries approximately 100,000 copies of one of the DNA molecules in the sample.

Reversible terminator sequencing

Nanopore sequencing

Single stranded polynucleotides can only pass single file through a hemolysin nanopore. The presence of the polynucleotide in the nanopore is detected as a transient blockade of the baseline ionic current. PA-pico-Ampere
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Google

You tube: Pyrosequencing nanopore sequencing 23 and me Personal Genome Project

Website:

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Four classes of DNA polymorphisms

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Single nucleotide polymorphism (SNP)

Single base-pair substitutions Arise by mutagenic chemicals or mistakes in replication Biallelic only two alleles Ratio of alleles ranges from 1:100 to 50:50 About 10 million human SNPs identified (23 and me assays 550,00 SNPs) (Chimpanzee and human have 35 million SNPs and 5 million indels) Most occur at anonymous loci Mutation rate of 1 X 10-9 per locus per generation Very few are thus new mutation in the species Useful as DNA markers 12

Fig. 11.3

Microsatellites
1 every 30,000 bp Repeated units 2 5 bp in length Mutate by replication error Mutation rate of 10-3 per locus per gamete Useful as highly polymorphic DNA markers
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Minisatellites
Repeating units 20-100 bp long Total length of 0.5 20 kb 1 per 100,000 bp, or about 30,000 in whole genome

Fig. 11.4

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Deletions, duplications, and insertions

Expand or contract the length of nonrepetitive DNA Small deletions and duplications arise by unequal crossing over Small insertions can also be caused by transposable elements Much less common than other polymorphisms
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RFLP: Restriction Fragment Length Polymorphism

HindIII HindIII 2 kb HindIII HindIII 5.7 kb HindIII

Ler Col
Probe:

HindIII digestion Electrophoresis Southern blotting Hybridization with the probe autoradiography
1kb ladder Ler

Col Het

2 1

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 SNP detection using southern blots

Restriction fragment length polymorphisms (RFLPs) are size changes in fragments due to the loss or gain of a restriction site

Fig. 11.6

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Example of RFLP
Restriction digestion GE Ethedium bromide stain Blotting Hybridization X-ray radiography

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CAPS (Cleaved Amplified Polymorphic Sequences) ie. RFLP detection by PCR

HindIII

Ler
HindIII

2 kb

HindIII

5.7 kb

HindIII

HindIII

Col

1kb ladder Ler

PCR HindIII digestion GE

Col Het

LCM
5

2 1

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Fig. 11.7

CAPS

Must have sequence on either side of polymorphism Amplify fragment Expose to restriction enzyme Gel electrophoresis e.g., sickle-cell genotyping with a PCR based protocol
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