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7.1 continued Techniques for Producing and Analyzing DNA

7.1 continued Techniques for Producing and Analyzing DNA. How do we sort out the DNA. DNA is chopped into many pieces How do we differentiate one piece from other??. Gel Electrophoresis. Technique used to separate charged molecules based on their size Acts like a molecular sieve.

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7.1 continued Techniques for Producing and Analyzing DNA

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  1. 7.1 continued Techniques for Producing and Analyzing DNA

  2. How do we sort out the DNA • DNA is chopped into many pieces • How do we differentiate one piece from other??

  3. Gel Electrophoresis • Technique used to separate charged molecules based on their size • Acts like a molecular sieve http://www.biotech.iastate.edu/ppt_presentations/html/Fingerprinting/StudentInstruction-gel/images/image08.jpg http://www.solve.csiro.au/1105/img/sieve-bloke.jpg

  4. DNA Preparation • Restriction enzymes digest DNA into smaller fragments of different lengths • Negatively charged dye and ethidium bromide are added • Different DNA samples are loaded into wells of the gel (agarose or polyacrylamide) http://www.oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg

  5. Gel is placed in a buffer and the DIRECT CURRENT power is turned on-NOTICE: negative and positive elctrodes.

  6. Gel Electrophoresis: Attraction Migration • Negatively charged DNA migrate towards positive end due to attraction • Smaller fragments experience less resistance and move further…blue dye makes DNA visible.

  7. Visualizing DNA Fragments • Gel is removed from buffer • Ethidium bromide attaches to the nitrogen bases in the DNA fragments and fluoresces under ultraviolet light http://www.answers.com/topic/agarosegel-jpg

  8. DNA fingerprinting or profiling: Traditional method-R F L P • Treat with restriction endonucleases • Separate fragments with gel electrophoresis • Do comparative analysis between samples-the bands on a gel are unique to each individual… Restriction Fragment Length Polymorphism

  9. DNA fingerprinting or profiling: More recent method-STR profiling • Using Primers and PCR, sections of the genome are amplified then separated by gel electrophoresis. • DNA sections are short repetitive sequences found at specific location in the genome. They are present in all but vary in length between individuals. • Longer fragments mean more, longer repeats…heavier masses=less movement in gel

  10. STR profiling produces a series of peaks that represent STRs of differing molecular mass and, therefore, differing lengths. Each individual has a unique series of peaks in their STR profile.

  11. How can we use Fingerprinting? • Forensic investigation of crime scene evidence • Post-mortem identification • Paternity Complete page 295 #7-12

  12. DNA SEQUENCING • Method for determining the nucleotide sequence-base by base-of a fragment of DNA. • The ultimate in DNA analysis!!!

  13. METHOD(S): MAXIM-GILBERT SEQUENCING -radioactively label single stranded DNA sequences -cleave at specific bases and separate with gel DIDEOXY SEQUENCING -DNA sequencing technique based on DNA replication using dideoxynucleosidetriphosphate

  14. P 295

  15. DNA is denatured into single stranded and primers are added to 3’ end Into 4 reaction tubes, add: • Single-stranded DNA with primer • DNA polymerase • Free nucleotides (dATP, dTTP, dGTP, dCTP) • One of four radioactively labeled dideoxy analogue (dd..)

  16. -Reaction tubes are loaded into wells and run through gel electrophoresis to separate the fragments.-Gels are exposed to x-ray film: autoradiography.

  17. The gel is read from the top to the bottom-longest to shortest fragment..this provides the complement of the original DNA sequence.

  18. 3’AGCCTAGAATTG 5’ synthesized strand original DNA 5′-TCGGATCTTAAC-3′

  19. So What?

  20. Human Genome Project-Used an automated version of dideoxy sequencing to sequence the entire human genome...identifying all the genes within.

  21. And the method gets better and better.... But why bother to keep improving? • Current automated techniques (2010) can sequence genomes with 1/12 the number of reactions it took in 2008...

  22. Oh the possibilities Medical advancements: • Individualized diagnosis and treatment based on individual genome • Cancer diagnosis and treatment • tumour profiling for individual care New technologies: • Site directed mutagenesis... • Ability to target specific sequences in genes and alter them to better understand structure and function.

  23. Page 300 Read through the section summary and complete the following... Review questions # 8-12

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