Microbiology Module 9

Part of microbial immune system.Patrol microbial DNA. Make DS break at specific sequences (GAATTC) in invading DNA and destroy it.

Microbes modify their own DNA so that it is immune to REs within their own cells

Each RE recognizes a specific 4-8 base pair palindromic sequence

REs are used to cut and paste (ligate) DNAs from different sources --> produces recombinant DNA

DNA is cloned when it is isolated from its original source and inserted into a vector that can be propagated in a host

Vectors are self-replicating DNAs (plasmids or phage) that also contain elements that allow manipulation and/or expression of cloned genes

DNAs are inserted into vectors, then vectors/inserts are propagated in host microbial cells (yeasts, bacteria)

Plasmids used to clone small pieces of DNA. Bacteriophage used to clone bigger pieces of DNA ( 9-25 Kbases)

Plasmid-derived vectors are introduced by transformation into artificially competent (CaCl2 or electroporation) microbial hosts

Phage or viral DNAs are packaged (in vitro) and then used to infect host cells

Origin of Replication (OriP) used to maintain high copy number (often 100s in a cell) or low, as required

MCS (Multiple Cloning Site) has multiple RE sites that are present only at this site. Used as cloning sites for genes

As only 1% of cells are competent for plasmid transformation, selectable markers and selective media are used to facilitate screening

Other elements: Gene expression, shuttle vector features

mRNA cannot be directly cloned, so functional mRNA (introns removed) from eukaryotic cells is first converted to cDNA using reverse transcriptase and dT primers

Resulting DS-DNA is then cloned by adding artificial RE sites to the ends and inserting into a vector

cDNAs from any source can be expressed as proteins when cloned into expression vectors containing proper transcription and translation genetic elemetns

Most widely used diagnostic technique in biological science

Amplifying via DNA replication the sequence of one particular fragment of DNA

Amplification of DNA is 10^6--10^9X for one round

Some substances inhibit thermophilic DNAPs and give false negatives

False positives (most often) because of extreme sensitivity of technique (Single cell's gene can be detected) and cross-contamination via aerosol

PCR'd genes can be cloned, sequenced, expressed

Template (can be any source of DNA) is heated to denature

Primers are short ssDNAs that bind at ends of denatured template (RE sites can be added) as temp is decreased

DNA is replicated at high temps using DNAPs from thermophiles and dNTPs

Repeat 20-30 cycles takes 1-3 hours

Currently these reactions are done with thermocyclers but field versions are being developed

Each sequencing machine can sequence 10^6 b/day. Provide lots of info. Can allow for microbial genome to be sequenced rather quickly

With shotgun procedures, whole genomes are randomly sheared and sequenced, then the overlapping sequences are assembled by a computer