Genomics

Complete Genomes in The Public DataBases

>100 Non-Eukaryotes

Eukaryotes:Leishmania 257 Kb 79 orfsPlasmodium falciparum I 947 Kb 205 orfs Plasmodium falciparum I 1,060 Kb 220 orfs Guillardia theta 551 Kb 464 orfsArabidopsis thaliana 115,428 Kb 25,498 orfs Oryza (sativa L. ssp. Indica) 420,000 Kb 50,000 orfsOryza (sativa ssp. Japonica) 420,000 Kb 50,000 orfsSaccharomyces cerevisiae 12,069 Kb 6,294 orfs Schizosaccharomyces pombe 14,000 Kb 6,824 orfsCaenorhabditis elegans 97,000 Kb 19,099 orfsDrosophila melanogaster 137,000 Kb 14,100 orfsMouse Musculus ~ 3,000,000Kb ~50,000 orfsHomo sapiens ~ 3,000,000Kb ~50,000 orfs

SEQUENCING (according to Sanger et al.)

A. Annealing and elongation

B. Termination

Manual Sequencing

4 lanes / sample

10 samples / gel

200-400 bases long

a g c t a g c t a g c t

1 2 3

Dye Terminator Sequencing

Sequencing

DNA templateprimersdNTPs

A

C

T

GA

C

A

G

T

T

GT

ddNTPs

C

G

T A

G

G C

G C T

G C T A

C G A T C C G T A A A

C G A T C C G T A A A

C G A T C C G T A A A

C G A T C C G T A A A

Dye Terminator Cycle Sequencing

Primer Walking

Shotgun Sequencing

Single Nucleotide Polymorphism (SNP)

• Single nucleotide polymorphisms (SNPs), i.e., single nucleotide base substitutions in gene sequences, account for 85% of the genetic variability in humans.

• This variability quite often accounts for differences in individuals' susceptibility to diseases and varying response to therapeutics.

• SNPs also have high utility in discovery of novel candidate genes through high resolution mapping

2. Purify PCR products/ Eliminate primers and dNTPs

SNaPshotTM Multiplex Kit Work Flow

1. Target Amplification

C

G

3. SNaPshot™ Reaction Kit

SNaPshotTM Multiplex Kit:Single Base Extension

ddGTPddCTPddUTPddATP

CCATGACTGATTCCNNNNNNAGCCTGGTACTGACTAAGGCNNNNNNN

Interrogation target

AmpliTaq® FSG

SNaPshotTM Multiplex Kit:Single Base Extension

ddCTPddUTPddATP

CCATGACTGATTCCNNNNNNAGCCTGGTACTGACTAAGGCNNNNNNN

Interrogation target

G

AmpliTaq® FS

Extends and Terminates Primer

C G A T C C G A A A A

PCR product

ACG CG GT

C G A T C C G A A A A

C G A T C C G T A A A

primer

CG

T A

A

C

T

G

ddNTPs

C G A T C C G T A A A

ACG CG GT

ACG CG GT

ACG CG GT A

A

T

T

SNaPshot reaction heterozygote

Sub

DNA micro-arrays

DNA Microarray (Filter)Oncogenes/Cell cycle Signal Transduction Apoptosis/DNA Repair

House Keeping

Transcrption Factors Receptors Growth Factors

Glass DNA Chip

Photolithography