Ch.10 Digital Analysis of DNA

Human Genome Project

An accurate sequence of the human genome was completed in 2003

• 2026: genome of 2287 animal species have been sequenced

General steps behind genome sequencing (4)

1. Fragmenting the genome

2. Cloning DNA fragments

3. Sequencing DNA fragments

4. Reconstructing the genome sequence from fragments

Methods of Fragmenting DNA (2)

1. Restriction enzymes (fragments at specific sites in DNA)

2. Mechanical shearing (random fragmentation)

Restriction Enzyme

Fragmennts DNA

specific sequence of bases anywhere within the genome + cuts sugar-phosphate backbones of both strands

• Recognition sits 4-8 bp

Mechanical forces

Fragmenting DNA

can break phosphodiester bonds at random locations

• passing DNA through thin needle at high pressure

• Sonication

Gel electrophoresis

movement of charged particles in an electric field

• Place gel in buffered aqueous solution and load DNA samples into wells in gel. Apply electric current. Stain w/ flourescent dye, photograph

• Smaller moves faster through

• DNA = neg charge (moves towards + charge)

Molecular cloning

to purify a specific DNA fragment away from all other fragments and make many identical copies of the fragment

Steps of cloning fragments (2)

1. Insert DNA fragments into cloning vectors to make a recombinant DNA molecule.

2. Transport recombinant DNA into living cell to be copied

Plasmid (3)

clones vectors; digest vector + DNA w/ restrict enzyme = comp sticky ends

1. Origin of replication

2. Restriction site(s) for cloning insert DNA

3. A selectable marker (e.g. antibiotic resistance)

Ligase (Plasmid vectors)

seal the phosphodiester backbones between vector and insert

Transformation

the process by which a cell or organism takes up foreign DNA

• Only the cells with plasmid will grow on media with ampicillin

• Ecoli only 0.1% are transformed

  
  

Genomic Library

Long-lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector

• each colony has diff recombinant plasmid

Sanger sequencing (chain termination method)

good for sequencing small pieces of DNA by using dideoxynucleotides (ddNTPs) that lack 3’-OH group that terminates DNA synthesis = nested array

• Each base has diff color (T,G,C,A)

Nested Array

DNA frags differ in length from the proceeding and succeeding fragment by one nucleotide

• tells # of bases in chain + last base

• smaller DNA frags move quickly to bottom

Automated DNA sequencing (Sanger Sequencing)

DNA fragments flow past a laser beam, the flouresent dye (used to detect ddNTP) creates peaks to comp strand, then computer reads 5’-to’3’

• Smaller frags on left (migrating first)

5 steps of the hierarchical strategy in Human Genome Project

• Drawbacks: labor-intensive, expensive, inefficient

1. Construct BAC (Bacterial Artificial Chromosome) genomic library

2. Identify sets of overlapping BAC clones and minimum tiling array for each chromosome

3. Shear DNA from each BAC separately to make smaller clones

4. Sequence DNA

5. Assemble sequences based on overlap

Shotgun Sequencing

Whole genome sequencing

• Problem: identical repetitive sequences can be misaligned

Paired-end sequencing (whole-genome shotgun strategy)

Shear genomic DNA into clones and sequence them from both ends based on overlap

• provides spatial information to help correctly assemble the genome

• unique sequences flanking the repeats to avoid repeat DNA