Lecture 27 - DNA Sequencing and Genomics

What is required for Sanger’s DNA sequencing reaction?

1. DNA polymerase

2. dNTPs (dATP, dTTP, dGTP, dCTP)

3. ddNTPs (ddATP, ddTTP, ddGTP, ddCTP)

4. Mg2+

5. DNA (template and primer)

What are the Sanger sequencing reactions?

1. 4 DNA replication reactions stop polymerization at A, T, G, or C

2. Results in DNA fragments that vary in size BECAUSE polymerization has prematurely stopped at every possible nucleotide

3. Different sizes of prematurely terminated DNA strands are used to determine the sequence of nucleotides

Fluorescent labeled dideoxy-terminators

All 4 reactions can be combined and terminating bases are distinguished by color

Scanning laser for sanger sequencing

No gels are required. Reaction sample is loaded onto a capillary tube and fluorescence identifies fragments as they move through the tube

Chromatogram

Laser detects sequence of bases based on color intensity

What are two requirements of DNA synthesis

1. Primer that provides a 3’ -oh

2. a template

High-throughput sequencing

Parallelizes the sequencing process and produces thousands or millions of sequences at once. Able to sequence a mix of DNA fragments and SKIP the cloning step

Genomics

The study of genomes of organisms

Bioinformatics

Acquiring, storing, manipulating, retrieving, and analyzing sequenced data in centralized databases

Subtractive hybridization

Used to identify genes expressed in one group of cells that are NOT expressed in a second group

BLAST

matches DNA to database

Homologue

Related gene with common ancestor

Paralogue

Homologue in the same species

Orthologue

Homologue in a different species

Why is protein sequencing better for identifying gene orthologues in distantly related species?

Protein sequence does not diverge as rapidly as nucleotide sequence in gene (cDNA)

Describe the hierarchical shotgun approach

1. Genome is broken into smaller pieces

2. These pieces were spliced into vectors (BACs)

3. Each piece was sequenced separately and then assembled using overlapping sequences from different pieces

What are reasons for the human genome not being fullt sequenced?

1. Centromeres and telomeres are highly repetitive and are difficult to sequence

2. Several regions contain multigene families that are difficult to disentangle with shotgun sequencing method

3. There are a few dozen gaps scattered around the genome

What are 3 types of key splicing?

1. Exon skipping

2. Intron retention

3. Alternate splice sites

Exon skipping

An exon is included or skipped

Intron retention

An intron is retained in the mRNA and often used to turn off gene expression

Alternate splice sites

Modifies the length of the exon