DNA-based information technologies

Understand basics steps/enzymes/features of DNA cloningKnow main types of cloning vectors used - pros & cons of eachUnderstand use of “probes” to identify DNA sequencesUnderstand use of expression plasmids to study gene productsUnderstand how site-directed mutagenesis is done and why it is useful to study proteinsKnow types of DNA libraries created & used

DNA cloning - separating a specific gene or DNA segment from a larger chromosome, attaching it to a small molecule of carrier DNA and then replication

5 general procedures of genetic engineering/recombinant DNA technology:

1. Cutting DNA at specific locations - restriction endonucleases

2. Selecting a small molecule of DNA capable of self-replication - cloning vectors

3. Joining two DNA fragments covalently - recombinant DNAs

4. Moving recombinant DNA from test tube to a host cell

5. Selecting or identifying host cells that contain recombinant DNA

RESTRICTION ENDONUCLEASES - protein enzymes that cleave the phosphodiester bonds that connect the nucleotide units in DNA or RNA at very SPECIFIC sites

These enzymes are mainly produced by bacteria where they degrade invading foreign DNA (bacterial DNA protected by methylation of its DNA); REs have been purified from these sources and are now available commercially

Most restriction enzymes recognize a specific sequence of 4-6 nucleotides in DNA and each will cut the DNA into discrete pieces known as restriction fragments

May need to purify DNA by gel electrophoresis/HPLC

Cloning vectors - plasmids, bacteriophage, BACs/YACs (HACs)

PlasmidsCircular DNA molecules that replicate separately from the hostIntroduced into bacterial cells by transformation or electroporationNaturally occurring plasmids ~5000-400,000 bp

Features of a plasmid:1. Origin of replication2. Antibiotic resistance for selection3. Unique restriction sites4. Small size facilitates entry into cell

Difficult to transform large plasmidsinto bacterial cellsDifficult to clone DNA segments >15,000 bp when plasmids are used as the vector

Cloning vectors - plasmids, bacteriophage, BACs/YACs

BacteriophageInfects bacteriaCan accommodate larger segments of DNA than plasmids

Features of bacteriophage:1. ~1/3 of its genome (48,502 bp) is nonessential and can be replaced with foreign DNA2. DNA is packaged into infectious phage particles only if it is between 40,000 and 53,000 bp long - this ensures the packaging of recombinant DNA only

Infect bacteria

Cloning vectors - plasmids, bacteriophage, BACs/YACs

Bacterial Artificial Chromosomes (BACs)Plasmids designed for the cloning for very long DNA segments (100,000 - 300,000 bp)

Features of BACs:1. Must have selectabel marker2. Must have very stable origin of replication

Use electroporation to get BACs into cells

Par genes - Assist in even distribution of plasmids to daughter cells at cell division

Low copy number plasmid - this limits the opportunities for unwanted recombination

Lac Z gene - -galactosidaseBlue-white colony screeningSubstrate - X-gal

Cloning vectors - plasmids, bacteriophage, BACs/YACs

Yeast Artificial Chromosomes (YACs)Eukaryotic organism for the cloning for long DNA segmentsMost used - S. cerevisiae (14 x 106 bp) - sequence knownEasy to grow and maintain

Features of YACs that allow them to be maintained as a eukaryotic chromosome in the nucleus:1. Yeast origin of replication2. Two selectable markers3. Specialized sequences (telomere & centromere) needed for stability and proper chromosomal segregation

Cloning vectors - YACs

Telomeres - sequences at the ends of chromosomes that help stabilizeYeast have 100 bp of imprecisely repeated sequences: (5’)-(TxGy)n; x~1, y~4, n~20-100Sequence lost here each round of replication - telomerase

Centromeres -DNA sequence that functions during cell division as an attachment point for proteins that link the chromosome to the mitotic spindleEssential for the equal and orderly distribution of chromosome sets to daughter cells

Genomic fragments are separated by pulsed field gel electrophoresisDNA fragments can be up to 2 x 106 bp

Stability of YAC clones increases with with size up to a pointInserts >150,000 bp stableInserts <100,000 bp are gradually lost

Cloning - specific DNA detection by hybridization

Sequence-based process for detecting a particular gene - use of probes

Cloning - specific DNA detection by hybridization

Design of probe??

Cloning - protein expression

Presence of correct sequence environment for eukaryotic DNA - expression vectors

Cloning - study of function of proteins using site-directed mutagenesis

From genes to genomes - creation of DNA libraries

DNA library - collection of DNA clones gathered together as a source of DNA for sequencing, gene discovery, or gene function studiesGenomic library - produced when complete genome of a particular organism is cleaved into thousands of fragments and all fragments are cloned by insertion into a cloning vector

Using probes, order clones in a library to identify overlapping sequencesSet of overlapping clones represents a long continuous segment of genome called a CONTIG

Known sequences in a library are called sequence-tagged sites (STS) and aid in genomic sequencing projects

From genes to genomes - creation of DNA libraries

Currently used libraries include genes that are expressed in an organismCreate cDNA from transcribed RNAs - clone into vector - creation of cDNA library

Aid for mapping of large genomes - cDNAs in a library are partially sequencedto produce a useful STS called an Expressed sequence tag (EST)

From genes to genomes - creation of DNA libraries

cDNA library made more specialized by fusing a reporter gene to cDNA sequenceGFP (green fluorescent protein)fused to allow study of location and movement of protein

From genes to genomes - creation of DNA libraries

Use of PCR to amplify specific DNA sequences