GENOMICS & SEQUENCING OF GENOMES

Genomics

the study of an entire genome(s)

Subfields of genomics

• functional genomics

• comparative genomics

Functional genomics

examines relationship between genotypes or sequences at any loci with the resulting phenotype

• ex: GWAS heart disease

Comparative genomics

align genome sequences between two or more group (e.g. species) to identify differences or similarities)

• ex: comparing wooly mammoth + modern elephant genomes

Medical genomics

identification of gene variants that make cancers aggressive

• sequence DNA from diff breast cancers, some w/ good prognosis & some w/ poor prognosis

• identify & compare mutations between the two groups

• compare genome from patient’s tumor to the known genomes

Personalized medicine

tailor treatment of disease to a person’s specific genome profile

Pharmacogenetics

identify drug treatments that correspond w/ a person’s genome profile

Human genome project

• done using the whole-genome shotgun approach

• create genomic library → sequence each clone

Sanger sequencing (dideoxy sequencing)

• sequence DNA using one relatively short piece of DNA at a time

  • still used today to sequence one or a few segments of DNA

• based on PCR amplification of the DNA you want to sequence

  • template can be linear or circular

• Taq polymerase adds nucleotides starting from a primer based on complementary sequences

Chain-terminating reaction

• Both dNTPs and ddNTPs are added to the reaction

  • Polymerase can add either dNTP or ddNTP equally well (doesn’t favor one over the other)

• Incorporation of ddNTP causes synthesis of that one new strand to stop 

  • Another base can’t be added to its 3’ carbon

• Each ddNTP is labeled a different fluorescent color

• Reaction products are a mixture of different length products, each with the last nucleotide labeled according to the identity of the base

Capillary Sanger sequencing

• reaction products are separated by capillary gel electrophoresis

• products come out the bottom of the capillary in size order

• laser at the bottom of the capillary excites fluorescence and the emission is detected

• order of fluorescence dictates order of bases

Chromatogram

• different colored peaks represent different bases

  • A = green

  • T = red

  • C = blue

  • G = yellow (black)

• read the order of the colored peaks to deduce sequence

Shotgun genome sequencing

1. extracted DNA from many individuals

2. cut DNA into small, overlapping fragments (called contigs)

• cut DNA using restriction digestions done in suboptimal conditions so that the R.E. didn’t cut at every site of DNA

3. made a genomic library from contigs

• each clone contains a different contig

• isolated each clone

4. sequenced each clone using Sanger sequencing 

• got sequence of each contig

5. reassembled contig sequences by puzzling together overlapping sequences

• created one continuous sequence for each chromosome

6. deposited info in NCBI database