Selected Techniques Part I

Transcription

1 1 Selected Techniques Part I Gel Electrophoresis Can be both qualitative and quantitative Qualitative About what size is the fragment? How many fragments are present? Is there in insert or not? Quantitative Exactly how big is the fragment? What is the concentration of the DNA being loaded into the well? Isolation and purification of DNA from gel.

2 2 Agarose vs. PAGE PAGE Polyacrylamide Gel Electrophoresis Agarose Large range Can separate DNA molecules that are many kilobases long. Poor resolution Cannot resolve single base pair differences. PAGE Narrow range Can separate DNA molecules up to several hundred base pairs. High resolution Can distinguish between DNA molecules that differ by as little as one base pair. Pulsed-Field Gel Electrophoresis Very long DNA molecules (30 50 Kb) cannot penetrate the pores of an agarose gel. DNA can be forced to snake through the agarose by applying a pulsed electric field. The orientation of the electric field changes and the DNA molecule reorients itself and makes it way through the pores. The larger the DNA the longer it takes to reorient when the field changes. Can be used to determine the size of an entire chromosome.

3 3 DNA Hybridization Hybridization base-pairing between complementary single-stranded polynucleotides from two different sources. Allows identification of specific sequences. One of the complimentary molecules is a probe. A probe is used to search for a specific sequence. The probe must be labeled so that it can be located once it has found its target sequence. Radioactive label (P 32 ) Fluorescent label

4 4 In Situ Hybridization Slice organism into thin pieces Incubate with radioactive single stranded DNA ssdna will bind to mrna Look for areas where your gene of interest is being expressed

5 5 Southern Blot Hybridization Localizes specific DNA molecules once they have been separated by electrophoresis. Restriction digest genome Soak in alkali to denature the DNA fragments. Transfer to a positively charged membrane creating an imprint or a blot. The membrane is incubated with probe DNA that is complimentary to your sequence of interest. Take picture of membrane using X-ray film.

6 6 Northern Blot Hybridization Identify a particular mrna in a population of RNAs Procedure is similar to Southern blot No need to digest mrna with enzyme since the molecules are short. The separated mrnas are transferred to a positively charged membrane. Probed with a radioactively labeled DNA sequence. Why do this procedure? Determine the amount of a particular mrna present in a sample. Compare levels of gene expression (transcription) between different tissues of an organism. Amount of hybridization is related to the amount of mrna in a sample. Western Blot Hybridization Used to detect protein expression Isolate cell protein Run on a denaturing PAGE Separates denatured (uncoiled) protein by mass. Transfer (blot) to a membrane Incubate with a specific antibody Look for presence of the protein Presence of protein indicates mrna transcription which indicates gene activity.

7 7 Western Blot Hybridization Advantages Detect end product (protein) Can detect post translational modification Disadvantages Difficult to create antibodies Not very sensitive.

8 8 Library Screening DNA Library population of vectors that each contains a different DNA insert. Digest genome with a restriction enzyme. Mix with vector cut with the same restriction enzyme in the presence of ligase. Genomic libraries Total genomic DNA Used for sequencing the genome. cdna library Generate sequences of DNA which code for protein. mrna is converted to cdna using reverse transcriptase. The cdna is then ligated into vectors. Transform bacteria with vectors and culture.

9 9 Library Screening Colony hybridization A labeled complimentary DNA probe is used to identify regions of an insert which contains a gene of interest. A cdna library will have thousands of different inserts. A single bacteria will give rise to a colony of bacteria with the same insert. A positively charged membrane is pressed on top of the culture dish. Imprints of the colonies is transferred to the membrane. The colonies on the membrane are washed with a probe containing a complimentary sequence to the gene of interest. The colony is identified on the membrane. Since the membrane is an image of the colonies on the culture dish the colony can be identified and the DNA purified. Library Screening

10 10 Library Screening Problems with bacterial plasmids for library screen. Transformation efficiency is relatively low. Insert size is limited ~25kb insert Given the size of the human genome (3x10 9 nucleotides), would require ~1000 plates. Phage Library Bacteriophage a virus which infects bacteria. Two sections to a bacteriophage Head region (contains DNA) Tail region (required to infect bacteria) Making a phage library Digest phage DNA with restriction enzyme Digest DNA of interest with restriction enzyme Ligate phage DNA and DNA of interest Incubate DNA with head portion, then tail portion Functional phage formed Each phage will have an extra piece of DNA

11 11 Phage Library Making a phage library continued Grow a lawn of bacteria Infect bacteria with phage Each phage has a different insert Phage infect bacteria producing more phage ~50,000 plaques (viral colonies) per plate.

12 12 Phage Library Phage library screening Place nitrocellulose filter on plate DNA sticks to nitrocellulose Denature DNA Incubate with labeled DNA DNA that is complementary will bind Place on film Look for spots of radioactivity Isolate phage from original plate

13 13 DNA Chip Array DNA Chip Array Large scale gene expression study Determine in which cells genes are expressed Purify mrna from an organism genome. Make cdna Add cdna to a specially coated glass slide. Glass slide is coated with poly-lysine which is positively charged DNA sticks to the slide. Each square in the chip corresponds to a different cdna. DNA Chip Array Add fluorescently tagged cdna from gene(s) of interest to see if they are expressed. Example: Determine muscle and neuronal gene activity in a nematode. Make a cdna chip with mrna from C.Elegans. Create a red fluorescently labeled probe for one gene of interest that is expressed in muscle cells Create a green fluorescently labeled probe for another gene of interest that is expressed in neuronal cells. Hybridize probes to chip Spots that fluoresce red correspond to the gene expressed in muscle cells. Spots that fluoresce green correspond to the gene expressed in neuronal cells. Spots that fluoresce yellow indicate that both genes are expressed.

14 DNA Chip Array Real Time PCR Real Time PCR or quantitative PCR method of simultaneous DNA quantification and amplification Purify mrna Reverse Transcribe into cdna Use Random Primers or dt prime. Perform PCR reaction Add a fluorescent probe. Contains a quencher and a fluorescent label. The quencher prevents the probe from fluorescing. As the PCR reaction proceeds the quencher is removed allowing the probe to fluoresce. A special type of thermal cycler detects the amount of fluorescence. 14

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16 16 Real Time PCR Measure the amount of DNA present as the PCR reaction proceeds. The more fluorescence the more DNA which is present. The more DNA the more initial mrna therefore the more the gene is expressed. Uses Study gene expression over time. Gene expression in response to environmental changes. Gene expression in response to medication Reporter Gene Assay Detect gene activity. Replace your gene sequence with a gene that codes for a protein which can be detected. Do not alter the promoter or enhancer part of the gene which is upstream to the altered sequence. The product the gene can be easily detected. Protein levels correspond with transcription rates. Types of Reporter Gene Assays Reporter gene B-galactosidase Reporter gene CAT Reporter gene Luciferase Reporter gene GFP

17 17 Reporter Gene-B- Galactosidase The enzyme B-Galactosidase cleaves X-gal. The product is dark blue Measure the level of blue color using a spectrophotometer. The blue color the more transcription of the gene. Reporter Gene-CAT Chloramphenicol Acetyl Transferase Enzyme that supplies cells with chloramphenicol drug resistance Expression measured by measuring the amount of neutralized chloromphenicol drug.

18 18 Reporter Gene - Luciferase Luciferase is an enzyme found in fireflies. Breaks down the substrate luciferin. As luciferin is broken down light is produced. Levels of light measured with a spectrophotometer. The more light the more transcription of the gene. Reporter Gene - GFP Green Fluorescent Protein Isolated from squid Protein glows green when exposed to UV light. Measure the amount of luminescence