3 September, 2004

Chapter 20

Methods: Nucleic Acids

Overview• Different DNA molecules have very similar biophysical properties but

can be separated by size using gel electrophoresis.• Restriction endonucleases cleave DNA molecules at specific sequences. • Hybridization can identify specific DNA molecules.• Cloning refers to the insertion of a specific DNA fragment into an

autonomously replicating vector like a plasmid.• Libraries are collections of vectors containing different cloned DNAs.• The polymerase chain reaction uses repeated replication to amplify

specific target sequences in vitro.• DNA sequence is determined using dideoxy chain termination.• Genomes can be sequenced by using short overlaps to assemble

individual clones into larger contigs.• Comparative genome analysis is one of the most informative areas of

molecular biology.

Gel ElectrophoresisNegatively charged DNA migrates toward the positive electrode.

Gel matrix retards fragments: smaller fragments move faster.

Polyacrylamide gels have high resolution over a short range, agarose gels have low resolution over a large range.

DNA can be detected by staining, autoradiography, or hybridization.

Pulsed-field Gel electrophoresis can resolve very large molecules.

DNA migrates as supercoiled > linear > relaxed circle.

RNA migration depends heavily on secondary structure.

Restriction Endonucleases

These proteins recognize short target sequences, often palindromic, and make double-stranded cuts.

Frequency of cut sites in random sequence is 1 / 4n, where n is the size of the site.

Restriction enzymes

http://www.dnai.org/b/index.html

Hybridization can be used to identify individual DNA molecules.

Under the right conditions, relatively short single-stranded DNA probe, radioactively labeled and present in excess will bind to a complementary DNA sequence, making it also radioactive.

Probes can by synthesized as radioactive or can be labeled enzymatically.

Fluorescent labeling is a less sensitive but less hazardous approach.

Hybridization• Southern Blot

• Northern Blot

• Microarray hybridization

Cloning

Libraries• Often individual clones must be isolated from libraries.

• Genomic

• cDNA

Colony / Plaque Hybridization

PCR

Site-directed Mutagaenesis

DNA Sequencing

DNA Sequencing

Genome Sequencing• Shotgun + Contig Assembly is the most efficient method of genome sequencing (better than the ordered-clone

approach).

• Paired-end reads assemble contigs into scaffolds.

Genome-wide analysis

In-silico:

•ORFs

•5’ / 3’ splice junctions.

•Promoters

ESTs are the gold standard for gene status.

Comparative Genome Analysis

Synteny Maps

Blast