Genotypic Microbiological Methods Can be used to determine genetic composition of organisms: Identify organisms (diagnostics) Identify distinct groups of organisms (taxonomy/systematics) by examining similarity between organisms Examine temporal or spatial variation of populations Study genes function and regulation Assess viability of cells

Restriction Enzyme Digestion –RFLP –PFGE DNA microarrays PCR based methods Overview of Molecular (DNA) Methods Combination of methods e.g. ribotyping DNA sequencing DNA “fingerprinting” Labeled Probes

DNA isolation Grow cells to stationary phase (for maximum yield) Lyse cells with heat, enzyme, or detergent Digest proteins with proteinase K enzyme Separate DNA from other molecules by solubility, charge, size etc..( methods vary) Commercially available methods Use centrifuge to pass cell lysate through filter, DNA binds to filter Add solvent to filter and centrifuge DNA out into clean tube

Gel Electrophoresis Gel made of translucent, porous matrix DNA samples added to wells in matrix DNA migrates at a rate inversely related to log10 of amplicon size - +

Gel electrophoresis EtBr binds to DNA as it travels through the gel EtBr fluoresces under UV light When viewed in the dark UV light, the DNA appears as bright bands Larger fragments of DNA remain near the top and smaller ones migrate to the bottom

Hybridization and Annealing of Nucleic Acids Complimentary sequences of ssDNA will bind together to form dsDNA Temperature at which dsDNA remains together depends on percent of matching and GC content Does not yield the DNA sequence of organisms, just the sequence similarity between organisms Total genomic hybridization can be used to estimate overall genetic similarity between organisms Oligonucleotide primers and probes can be designed to detect and ID genes

Labeled Probes Oligonucleotide-short piece of DNA Complementary-has corresponding nucleotide base sequence (ATCG- TAGC) Target- specific region of organism’s DNA to be probed Labels- a molecule that is attached to the probe and can be observed by some direct (fluorescent) or indirect means (immunodetection) ….ATTGCGGCTCCATACGTAACGTACGGACTGACTTAGCT…….. TATGCATT TAACGCCGAGGTATGCATTGCATGCCTGACTGAATCGA

RFLP RFLP-restriction fragment length polymorphisms DNA RE cuts wherever it recognizes specific site Organism A Organism B Gel electrophoresis _ +

Separation and visualization DNA fragments Large pieces of DNA Small pieces of DNA L L _ +

Ribotyping DNA EcoRI cells Transfer to nylon membrane (Southern Blot) 1% Agarose Gel Bind labeled 16S rDNA Probe - + Gel electrophoresis Anti-probe Ab and enzyme-linked color reaction

PFGE (Pulse Field Gel Electrophoresis) Agarose gel

Repeatability and Discrimination Trial 1Trial 2

PCR polymerase chain reaction Invented by Kary Mullis in 1983 Now widely used for many types of scientific research and medical diagnostics Works by amplifying target region of DNA using: – synthetic oligonucleotides called primers –Taq polymerase enzyme –Temperature cycling

Concept Amplify small quantities of DNA by in vitro DNA replication Target DNA PCR Copies of Target DNA (amplicons)

Generalized PCR cycle repeated ca. 40 times 94 degrees Celsius- denaturation Ca degrees- primer annealing 72 degrees Celsius- extension Target sequence Taq

Primers 3’…GTATTATGGTATGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA… 5’…TATCGAACGGAGACTTACTCTTATACCGTGGTAGCTTTGTAATGATATT… 5’TACGAACGG 3’CCATCGAAA Specificity of PCR depends on the sequence to which they bind primers

Extension (polymerization) 3’…GTATTATGGTATGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA… 5’…TATCGAACGGAGACTTACTCTTATACCGTGGTAGCTTTGTAATGATATT… 5’TACGAACGG 3’CCATCGAAA Taq

PCR Primers anneal to both strands of target sequence New strands of DNA are added (5’ to 3’) from the primers In subsequent cycles, primers can bind to amplicons in addition to the original DNA

1 st Cycle 2 nd Cycle Single copy of dsDNA target 3 rd Cycle Amplicons increase exponentially with each cycle

Factors that influence specificity Stringency of conditions –Degree of primer sequence match to target sequence –Primer length –Annealing temperature Uniqueness of target sequence –Primers will bind wherever there is a complementary target sequence

Factors that influence sensitivity Presence of necessary components –Taq –dNTPs –Magnesium –Target sequence –Primers Presence of inhibitory chemicals Primer hairpins and self dimers

Can be used to generate qualitative or quantitative data L Positive Charge Negative Charge L

Real Time PCR Time fluorescence Can be used to estimate the starting concentrations of DNA

Reverse Transcriptase PCR RNA converted to cDNA by reverse transcriptase enzyme cDNA used to perform PCR Used to detect specific RNA viruses Used to test viability of cells

DNA sequencing DNA usually in the form of PCR amplicon One strand at a time Most thorough method of studying variation in DNA and assessing DNA similarity Relatively expensive and time consuming (however, this is constantly improving) Can now sequence entire genomes of organisms

Extension (polymerization) 3’TAGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA… 5’ATCGAACGGAGACTTACTCTTA Taq CA A T G C A T A G T T A 3’TAGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA… Taq 5’ATCGAACGGAGACTTA dNTPs are randomly- incorporated into new strand until a ‘stop’ is added

Possible fragments A G C T T A G T If there is contradictory info, it will be read as ‘N’

Sequence Trace

DNA microarrays (Gene Chips) ~1cm Microarray Chip containing thousands of blocks Each block is affixed with numerous copies of a nucleic acid sequence Labeled complementary nucleic acid binds and stimulates chip