Genomic DNA Extraction of Coal Isolates

Polymerase Chain Reaction and Gel Electrophoresis:Theory and ApplicationKei-Anne BaritugoDepartment of Environmental EngineeringMyongji UniversityMS/PhD Student

Brown, T. A,. Gene Cloning And DNA Analysis. Oxford: Blackwell Science, 2001. Print.

Contents: IntroductionPCR Theory and conceptsTypes & ApplicationsGel ElectrophoresisTheory and conceptsApplicationPaper Review

Polymerase Chain Reaction

http://passel.unl.edu/pages/informationmodule.php?idinformationmodule=1130447283&topicorder=6&maxto=9&minto=1https://www.uvm.edu/~gdrusche/Classes/GEOL%20295%20-%20Geomicrobiology/Lectures/Lecture3.ppthttp://www.slideshare.net/sujathar23/pcr-and-types3Polymerase Chain Reaction

http://en.wikipedia.org/wiki/Applications_of_PCRhttp://www.slideshare.net/sujathar23/pcr-and-types4Polymerase Chain ReactionType of PCRModificationApplicationColony PCRMultiple DNA samples are used

DNA primers are specific to sequence of gene insert so only gene of interest is amplifiedScreening of microbes for correct DNA insertNested PCRTwo pairs of primers are used

1st pair amplifies region flanking gene of interest 2nd pair binds to inner region of 1st ampliconVery low probability of nonspecific amplificationInverse PCRAmplification of DNA with only one known sequence (involves restriction and ligation)

Amplification of unknown sequence from a known template

Used in identifying flanking sequence of genetic inserts

Identification of sites where retrovirusesintegrate into genomic DNAMultiplex PCRUse of more than one pair of primersGeneration of a lot of gene of interests from one genome sampleLong PCR (5kb instead of normal 10kb)Use of Pfu polymerase (Pyrococcus furiosus) instead of Taq polymerase (Thermus aquaticus)DNA sequencing of genomes or long DNAhttp://cdn.intechopen.com/pdfs-wm/37264.pdfhttp://www.slideshare.net/sujathar23/pcr-and-types5Polymerase Chain ReactionType of PCRModificationApplicationReverse Transcriptase PCRrtPCR

Qualitative detection of gene expressionTemplate is RNA > complementary DNA transcripts by using reverse transcriptase Gene Expression ProfilingIdentification of sequence of an RNA transcriptReal-time PCR qPCR

Quantification of DNASimultaneous amplification and detection or quantification of DNA target DNA molecule

+ nonspecific fluorescent dyes that intercalate w/ any dsDNA

+ sequence specific DNA probes made up of oligonucleotides labelled w/ fluorescent reporter permitting detection after hybridization probe binds to complementary sequenceDiagnostic research for infectious disease, cancer and genetic mutations

Assessment of water quality

Quantification of gene transcription

Clinical quantification and genotyping of Hepa B virusQuantification = degree of infection copy of viral genome / unit of tissueAsymmetric PCR2 primers are used 100:1 ratio after 20-25 cycles of amplification one primer is exhausted so single stranded DNA is produced in next 5-10 cyclessynthesis of singlestranded DNA

useful for sequencing or DNA probe preparationshttp://www.slideshare.net/sujathar23/pcr-and-typeshttp://en.wikipedia.org/wiki/Variants_of_PCR6Gel Electrophoresis

https://www.google.co.kr/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&sqi=2&ved=0CCoQFjAC&url=http%3A%2F%2Fwww.sunyjcc.edu%2Fhurisuri%2Fsites%2Fdefault%2Ffiles%2FPCR_and_Gel_Electrophoresis.ppt&ei=8cpyVZLnNaG2mAXiwoM4&usg=AFQjCNHVs4tN9_-_S7Htpud22Vsa4zOdrA&sig2=HfDh-rEvNWw-Hvv6luJPgg&bvm=bv.95039771,d.dGY&cad=rja7

Gel Electrophoresis

http://www.slideshare.net/harshit172/agarose-gel-electrophoresis-25523393

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Cobain/method.html8Review Article:Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor

Marc Rehfuss, James UrbanSystematic and Applied Microbiology 28 (2005) 421429

Methodology:Isolation of Bacterial StrainsMethodology:Morphological, Physiological & Biochemical Characterization

Confirmation of biolog reactions were checked with growth or lack of growth on M9 agar w/ same C-source as Biolog plates

http://www.iul-inst.com/images/GN2.pdf11Methodology:Extraction of DNA and 16S rDNA analysis

12Methodology:Extraction of DNA and 16S rDNA analysis

13Results and Discussion

14

Conclusion