Methods for Working with DNA and RNA

Transcription

1 Methods for Working with DNA and RNA 1. Gel electrophoresis A. Materials: agarose (large DNAs) vs. acrylamide (high resolution, DNA sequencing) B. Separated by its sieving property and charge: both are proportional to size of DNA C. Large DNAs are resolved by pulse field gel electrophoresis 1 min in forward direction and 15 seconds in reverse direction figure D. Visualize DNA by staining with such things as ethidium bromide intercalates into DNA and fluoresces, detection limit is near ng

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3 Methods for Working with DNA and RNA 2. Enzymes A. Restriction Endonucleases - i. originate from a host-specific modification a. host restriction endonuclease b. host modification methylase ii. three types of endonucleases a. type I and III are single polypeptides with both endonuclease & methylase activity b. type I cuts at least 1000 bps away from recognition site and type III within ~24-26 bps c. type II has separate endonuclease and methylase activity, and cuts at the specific recognition site

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5 Methods for Working with DNA and RNA 2. Enzymes A. Restriction Endonucleases - i. originate from a host-specific modification a. host restriction endonuclease b. host modification methylase ii. three types of endonucleases a. type I and III are single polypeptides with both endonuclease & methylase activity b. type I cuts at least 1000 bps away from recognition site & type III within ~24-26 bps c. type II has separate endonuclease and methylase activity, and cuts at the specific recognition site d. sticky ends vs blunt ends

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7 Methods for Working with DNA and RNA 2. Enzymes A. Restriction Endonucleases - i. originate from a host-specific modification a. host restriction endonuclease b. host modification methylase ii. three types of endonucleases a. type I and III are single polypeptides with both endonuclease & methylase activity b. type I cuts at least 1000 bps away from recognition site and type III within ~24-26 bps c. type II has separate endonuclease and methylase activity, and cuts at the specific recognition site d. sticky ends vs blunt ends B. Restriction Mapping and RFLP mapping i. restriction cut sites are physical reference points on a DNA molecule ii. Restriction Fragment Length Polymorphisms: a. DNA fingerprinting in criminal cases b. genetic screening for particular diseases

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11 Methods for Working with DNA and RNA 2. Enzymes C. Exonuclease i. 3' exonuclease ii. 5' exonuclease D. DNA ligases E. DNA polymerases F. Phosphatases G. Polynucleotide kinases H. Still more

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13 Methods for Working with DNA and RNA 3. Southern/Northern Blotting Purpose: is to identify the presence of a particular DNA (RNA) sequence in you sample.

14 modes of detection: primarily radioactivity or chemilluminescence

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17 Type of Blot Identify Probe or manner of detection Southern DNA using DNA or oligonucleotide probe Northern RNA using DNA or oligonucleotide probe Western protein using specific antibodies, electrophoretic transfer membrane, block membrane with BSA or milk protein Far Western protein for detecting one protein interacting with another protein South Western protein using a radiolabeled DNA probe

18 Methods for Working with DNA and RNA 4. DNA Micro Chip Arrays- Genomic wide analysis A. A thousand different DNAs are immobilized onto a glass slide using the same technology used by computer chip manufacturers B. Fluoresecently tag total cellular mrna from wild type cells and anneal to immobilized DNA C. Quantitate the total amount of each individual mrna by the amount of fluorescence at any given position in the slide, sensitivity ranges from mrna/cell D. Can determine the effect of one gene product on the genome wide expression of mrna by isolating mrna from deletion strain missing that gene or from a strain with a temperature sensitive strain.

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20 Methods for Working with DNA and RNA 4. DNA Micro Chip Arrays- Genomic wide analysis A. A thousand different DNAs are immobilized onto a glass slide using the same technology used by computer chip manufacturers B. Fluoresecently tag total cellular mrna from wild type cells and anneal to immobilized DNA C. Quantitate the total amount of each individual mrna by the amount of fluorescence at any given position in the slide, sensitivity ranges from mrna/cell D. Can determine the effect of one gene product on the genome wide expression of mrna by isolating mrna from deletion strain missing that gene or from a strain with a temperature sensitive strain.

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23 Methods for Working with DNA and RNA 4. DNA Micro Chip Arrays- Genomic wide analysis A. A thousand different DNAs are immobilized onto a glass slide using the same technology used by computer chip manufacturers B. Fluoresecently tag total cellular mrna from wild type cells and anneal to immobilized DNA C. Quantitate the total amount of each individual mrna by the amount of fluorescence at any given position in the slide, sensitivity ranges from mrna/cell D. Can determine the effect of one gene product on the genome wide expression of mrna by isolating mrna from deletion strain missing that gene or from a strain with a temperature sensitive strain.

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27 Methods for Working with DNA and RNA 4. DNA Micro Chip Arrays- Genomic wide analysis A. A thousand different DNAs are immobilized onto a glass slide using the same technology used by computer chip manufacturers B. Fluoresecently tag total cellular mrna from wild type cells and anneal to immobilized DNA C. Quantitate the total amount of each individual mrna by the amount of fluorescence at any given position in the slide, sensitivity ranges from mrna/cell D. Can determine the effect of one gene product on the genome wide expression of mrna by isolating mrna from deletion strain missing that gene or from a strain with a temperature sensitive strain.