Quiz 3 Flashcards
Southern Blot
Detects fragments of DNA that contains a specific sequence. DNA is cut with endonuclease. Fractioned by size with agarose gel electrophoresis. DNA is denatured and transferred to a nylon or nitrocellulose membrane using capillary action. Radioactive probe and washed. Xray visualizes those attached with a probe.
PCR
Amplifies minute quantities of DNA. Requirements include (1) a small sample of DNA with known sequence (2) synthetic oligonucleotides complementary to the target DNA, serving as primers and need to be present in excess (3) dNTP (4) Taq Polymerase (5) pH buffer and salts.
Microsatellite Repeats
Stretches of repeated di- or tri- nucleotides. The number of copies determines the overall microsatellite and fluctuates among individuals. DNA fingerprinting.
SNP- Single Nucleotide Polymorphism
Single nucleotide differences that do not alter restriction sites so cannot be distinguished with a Southern Blot. Detected by (1) amplify using PCR (2) divide DNA into two parts (3) denature DNA and hybridize it with a oligos representing the different SNP sequences (4) detect the double stranded product using various techniques like gel electrophoresis, HPLC, or fluorescence-based methods.
Sanger-dideoxy Sequencing
Uses ddNTP’s to terminate polymerization of nucleotide chains catalyzed by DNA polymerase. They lack the -OH group for elongation so the chain terminates. The products are denatured and run side-by-side on a gel. It can be read from the bottom up. Used to be radioactively labeled with phosphate to read. Now, the reactions can be done all at the same time using a fluorescently labeled probe with the products passing through a detector.
Massively Parallel DNA sequencing
A recent method in DNA sequencing that can read 75-100 bp of DNA up to 200 DNA sequences at the same time. DNA is fragmented using acoustic waves. Illumina adaptors are added and the DNA is size fractioned. A band of 200-300 bp is excised. Size-selected adapter-ligated DNA is used as template in a PCR to add P7/P5. Library is washed and DNA undergoes bridge amplification. Sequencing primer is anneal and base added one at a time. Flow cell is imaged.
Reverse Transcriptase PCR (RT-PCR)
Used to measure mRNA levels. Messenger RNA is the template. (1) anneal oligo-dT primer to the poly(A) sequence at the 3’ end of the mRNA (random primers and gene specific primers can also be used). (2) synthesis of first cDNA using reverse transcriptase. (3) PCR. More abundant mRNA will amplify more quickly. We can evaluate gene expression regulation.
Microarray
Measures relative changes in RNA levels. It can measure many at a time. RNA is measured from two sources and fluorescently labeled. It is hybridized to the microarray. Ratio of two dyes is calculated for each spot.
Microarray Comparative Genomic Hybridization
Using DNA instead of mRNA to interrogate regions of the genome for duplications or deletions.
Knock-Out
Gene disruption or loss of function mutation. Scenario in which a gene is disrupted such that it no longer produces a functional product. Insertion of stop codon, insertion of foreign gene within the targeted gene, or the complete deletion of a gene.
Knock-In
Targeted mutation. Mutation is introduced without disruption of other parts of the gene. Mis-sense mutation so that a mutant protein will be expressed from the targeted gene. It can also replace a mutant gene by knocking in the wild type.
Construction of DNA for Knock-In/Out
Generally contain the following (1) homologous region to the gene to be knocked out (2) region of dissimilarity to induce the knockout (3) positive and negative selection.
Positive Selection
Resistance to neomycin, puromycin, hygromycin to select for only those cells with the new DNA incorporated and expressed.
Negative Selection
Inserting a thymidine kinase gene that will only be taken out if it is inserted into the correct location. If this gene remains, ganciclovir will kill the cells, thus eliminating the ones that have an insertion in the incorrect location.
Chimeric Mice
Correct cells are injected into the blastomere of an embryo. They will be made from animals of two colors for identification. The resulting animal should be a mix of two colors. Mice then are bred for a “true-breeding” line.
True Breeding Line
Progeny must be screened for the mutation. If a black chimeric mice results, you proceed. The black mouse could be heterozygous or not mutated at all. Coat color is a separate gene so a Southern Blot or PCR can detect a gene in any black mouse. The mutation can then be introduced into different mouse strains. It can be combined with other genes or bred to homozygosity.
Human Embryonic Stem Cells (ES)
Derived from discarded embryos. They retain pluripotency. They express histocompatibility locus antigens (HLA) and may harbor latent tumorigenic properties.
Induced Pluripotent Cells
Generated from many cell types that were thought to be permanently differentiated. Small molecules and miRNA are thought to induce them.
Example of Gene Correction
Sickle Cell Anemia in Mice Cells
- Cells removed from living animal from the tip of the tail. Grow in culture dish.
- The cells are infected with viruses expressing the pluripotency factors and selection for ES like cells occurs.
- iPS clones are identified and characterized, one is selected for gene correction using a “knock-in” vector designed to replace the hemoglobin beta s gene with wild type.
- Clone with successful gene replacement is selected and screened.
- Induced to differentiate into blood cells.
- Transplant the normal blood stem cells, gene corrected and perfectly matched to the HLA.
Spermatogonia
Undifferentiated stem cells of the germ line. Abundant in seminiferous tubules. High rate of mitotic activity, which is thought to account for the accumulation of new mutation.
