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DNA-based information technologies
Understand basics steps/enzymes/features of DNA cloning
Know main types of cloning vectors used - pros & cons of each
Understand use of “probes” to identify DNA sequences
Understand use of expression plasmids to study gene products
Understand how site-directed mutagenesis is done and why it is useful to study
proteins
Know 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)
Plasmids
Circular DNA molecules that replicate separately from the host
Introduced into bacterial cells by transformation or electroporation
Naturally occurring plasmids ~5000-400,000 bp
Features of a plasmid:
1. Origin of replication
2. Antibiotic resistance for selection
3. Unique restriction sites
4. Small size facilitates entry into cell
Difficult to transform large plasmids
into bacterial cells
Difficult to clone DNA segments >15,000 bp
when plasmids are used as the vector
Cloning vectors - plasmids, bacteriophage, BACs/YACs
Bacteriophage
Infects bacteria
Can 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 DNA
2. 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 marker
2. 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 - b-galactosidase
Blue-white colony screening
Substrate - X-gal
Cloning vectors - plasmids, bacteriophage, BACs/YACs
Yeast Artificial Chromosomes (YACs)
Eukaryotic organism for the cloning for long DNA segments
Most used - S. cerevisiae (14 x 106 bp) - sequence known
Easy to grow and maintain
Features of YACs that allow them to be maintained as a eukaryotic chromosome in the nucleus:
1. Yeast origin of replication
2. Two selectable markers
3. Specialized sequences (telomere & centromere) needed for stability and proper chromosomal
segregation
Cloning vectors - YACs
Telomeres - sequences at the ends of
chromosomes that help stabilize
Yeast have 100 bp of imprecisely repeated
sequences: (5’)-(TxGy)n; x~1, y~4, n~20-100
Sequence 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 spindle
Essential for the equal and orderly distribution of
chromosome sets to daughter cells
Genomic fragments are separated by
pulsed field gel electrophoresis
DNA fragments can be up to 2 x 106 bp
Stability of YAC clones increases with
with size up to a point
Inserts >150,000 bp stable
Inserts <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 studies
Genomic 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 sequences
Set of overlapping clones
represents a long continuous
segment of genome called a
CONTIG
Known sequences in a
library are called sequencetagged 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 organism
Create cDNA from transcribed RNAs - clone into vector - creation of cDNA library
Aid for mapping of large genomes
- cDNAs in a library are partially sequenced
to 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 sequence
GFP (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