Methods that do not require growth in laboratory PCR

Transcription

1 Methods that do not require growth in laboratory PCR Nucleotide sequencing MLST/MLVST Profiles

2 Microarrays

3 Polymerase Chain Reaction [PCR] Use to find a rare sequence in a pool of many different sequences Amplifying an ancient DNA sample

4 Molecular photocopying sequence PCR assay goes between cycles; produces 10, ,000 s copies of sequence of interest Some primers can be universal for PCR such as bacterial 16S and other conserved genes Some primers are more specific for divergent genes

5 PCR products Use as a probe for Southern blot, in situ labeling Cut to give RFLP pattern use for typing Sequence determined for bacterial species identification, use in MLST/MVLST typing Prepare cdna from genomic RNA, mrna: make DNA copy of RNA genome

6 PCR products Used to determine genome organization of bacteria, viruses, parasites, plants, animals, man Bacterial Chromosome Used to determine composition of satellite DNA, mitochondrial DNA, plastid DNA, plasmids, specific genes, whole chromosomes, sequences generated can be used for typing Human Chromosome

7 Real time PCR [qpcr] Machine that allows both detection and quantification of copy # as PCR copies accumulate at the same time Determine # of copies in a sample Not generally used for typing RT-PCR (reverse transcription-polymerase chain reaction) Determine level of mrna expression: most sensitive method for mrna diction and quantitation Not generally used for typing

8 Advantages of PCR Provides great flexibility in working with unculturable microbes (HPV, Hepatitis C) and microbes that require growth in cell culture/whole animals Allows rare sequences to be amplified for further work Used for sequencing, MLST, MLVST typing, other types of typing Some standard protocols and controls exist Used to understand HPV infections and make the vaccine

9 Disadvantages of PCR Easy to contaminate PCR assay Needs expensive machine Needs highly trained technician Needs to separate where assay is mixed from machine and where DNA is made Need to have good controls Different brands of machines may required different assay conditions Taq enzyme is expensive

10 Nucleotide sequencing 1. Any organism with DNA or RNA can be sequenced 2. Look at variable regions of genes which mutate rapidly 3. Cost and time need to perform continues to decrease; now all automated Currently $2-6/rxn locally New machines can do 96 well plates in 8 h ~$200/plate 4. PCR now used to generate information: the 4 bases are tagged With different florescent tags 5. Larger fragments (~1 kb) can now be sequenced in some systems Locally can get ~ kb 6. Variety of programs look at similarity between > 2 sequences and Produce dendograms: different programs may give different relationships 7. Software limitations limits the ability to compare entire genomes genomes are broken up for comparisons Software and hardware are not yet able to compare whole chromosomes of anything larger than a small virus 8. Many of the software programs such as NCBI BLAST (free) are old not that user friendly for large sequences or multiple comparisons

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13 DNA Sequence 1 gagtgtgtag ctttgctcgt aaaagcttag aaacctttga acgaaaggga taatgaaagc 61 cttgattgca aggctttttg ctttatggtg ggtaagtatc agagtgagaa aatttttgga 121 atgagtagaa gtgatagcta gaaattatca gtttctattt ccatttaccc tgtgggtacg 181 tgtttgtttc cattgaaagg agtttgtggg aatagaaatg tacccacctt gtttgaatca 241 agtgaagtgt agttgaagga aatctgttga aagcaatact tcattttacc gaataagtaa 301 taatttaggc aacttcaaat cgattaaaaa aaactatttt aaaggttaag agtagacaaa 361 aattgtccac tcttttttgc aaactcaatt tatcaataaa tgaaatgagg gaatgtaaaa 421 tgaaatattt tgaggttgag ttagaaaatc ctgatgaatt tttaaaacta caaacagaag 481 attttgtgaa agctaatcgc ttgctactaa ggaagataat ccagagcgtt acagtctatg 541 aagaaaactt cgtcatatcc tttaaatctg gcatcgaatt ggaagtatga gtctcattcc 601 ataactttta tattgaacat atcatcttgt tgtgttatac tataaattga tataaacaaa Generate Amino Acid sequence MRECKMKYFEVELENPDEFLKLQTEDFVKANRLLLRKIIQSVTV YEENFVISFKSGIELEV Each letter represents different amino acid

14 Sequencing Used for: Viruses: HPV, Hepatitis C, HIV Bacteria: M. tuberculosis drug mutations Bacterial quinolone resistance Changes in penicillin binding proteins (PBP) Identify differences in toxin genes Serotyping Multilocus sequence typing (MLST) Multilocus virulence typing (MLVT) Drug resistance in yeast & parasites Man: Look nucleotide polymorphisms (SNAP typing) What is used to describe clonality in genes from bacteria, viruses (DNA vs RNA), and parasites differs

15 Advantages of Sequencing 1. Any organism with DNA/RNA can be sequenced 2. Sequence chromosomes, plasmids, mitochondrial DNA etc 3. Very discriminating since it identifies a) single base pair differences which may or may not translate into amino acid differences b) insertions, deletions and/or sequence rearrangements 4. Do not need to grow the organism 5. Need to know nothing about the organism if using Standardized primers 6. Price and time required to do is coming down 7. VERY VERSATILE, POTENTIAL NOT FULLY DEVELOPED

16 Disadvantages of Sequencing 1. Equipment and reagents are expensive and highly trained technicians required to do the work and analyze the data 2. Sequencing normally done at centers which are not always local 3. HANDLING LAREG DATA SETS REQUIRES TRAINING; BETTER HARD AND SOFTWARE NEEDED 4. How to interpret results is not standardized for most organisms and differs between DNA, and RNA viruses, bacteria, parasites, yeast and other eukaryotic organisms 5. No standards 6. Sequencing usually done in both directions and often both DNA strands which requires significant amount of data management after the data is generated before comparison can be done