Term
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Definition
| Pieces of DNA, from different origins, ligated to form one piece of DNA |
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Term
| restriction endonucleases |
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Definition
cut double-stranded DNA at specific palendromic sequences. In cutting the DNA, they leave either 5’ or 3’ overlapping ends (also called sticky
or staggered ends) or blunt ends. |
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Term
| How to see if restriction digest worked |
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Definition
| Separate by size. DNA fragments can be separated by agarose or acrylamide gel electrophoresis. DNA is negatively charged so the fragments migrate toward the (+) electrode, with smaller fragments moving faster than larger fragments of DNA. The DNA can be visualized by adding the dye ethidium bromide to the gel; the dye intercalates with the DNA and causes the DNA to fluoresce when illuminated with UV light. Smaller fragments migrate faster |
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Term
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Definition
| used for making genomic libraries |
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Term
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Definition
| used for making cDNA libraries and subcloning |
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Term
| essential properties of cloning vectors |
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Definition
Foreign DNA can be easily inserted, and vectors containing foreign DNA can be selected and distinguished from vectors which did not incorporate foreign DNA.
Replicate autonomously in host cell.
DNA is easily separated and purified from host DNA. |
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Term
| common features of vectors |
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Definition
| 1) antibiotic resistance; 2) unique endonuclease restriction sites for inserting (cloning) foreign DNA (in pUC, this is a polylinker cloning sequence), and 3) color selection with pUC vectors to help distinguish vectors that have successfully ligated a foreign DNA fragment from those that haven’t (blue color = no insert; clear/white color = positive for insert). |
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Term
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Definition
| used to facilitate cloning of foreign DNA. These enzyme sites are unique (present only 1 time, in this location, in the entire vector). |
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Term
| how to place unknown DNA into vector |
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Definition
| Ligate unknown DNA with vector, both cut with same RE. Transform bacteria so ligated vector enters cells and replicates. Select for bacteria which contain plasmids (antibiotic resistance and color-selection) |
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Term
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Definition
Cosmids are cloning vectors that
can incorporate large amounts of
foreign DNA (up to 45 kb). One
can use antibiotic resistance in the
selection of bacteria containing
cosmids. However, cosmids enter
bacteria through infection
following packaging into phage
Heads (not by transformation, like plasmids). |
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Term
| problems associated with cosmids |
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Definition
| Concatenation of the vector (cosmid-cosmid ligation), Insertion of multiple foreign DNA pieces, Instability of foreign DNA sequences, Differential growth of clones |
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Term
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Definition
Yeast Artificial Chromosomes; 100-500 kb; have been used to generated genomic
libraries because of their ability to insert large amounts of foreign DNA. They also confer antibiotic resistance when they are in bacteria. |
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Term
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Definition
bacterial artificial chromosomes; ~150 kb; have been used to generated genomic
libraries because of their ability to insert large amounts of foreign DNA. They also confer antibiotic resistance when they are in bacteria. |
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Term
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Definition
p1 phage artifical chromosomes; ~150 kb; have been used to generated genomic
libraries because of their ability to insert large amounts of foreign DNA. They also confer antibiotic resistance when they are in bacteria. |
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Term
| Structure of bacteriophage |
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Definition
| head contains the genetic material (DNA), a tail, and a tail fiber through which the DNA is passed through once the phage attaches to the bacterium. A single bacterium is only infected by a single phage, and a single phage can only infect a single bacterium. |
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Term
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Definition
| contain all of the DNA within an organism, including exons and introns, promoters, 5’ and 3’ flanking sequences, and intergenic regions. |
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Term
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Definition
| a reflection of the mRNA (genes) that is expressed in a particular tissue/organ at a specific developmental time |
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Term
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Definition
Isolate poly A+ mRNA.
Anneal a primer to the mRNA (often an oligo dT primer).
Make the first cDNA strand using mRNA as the template, a primer which is bound to the mRNA, dNTPs, and reverse transcriptase.
Synthesize the second cDNA strand using DNA polymerase.
RNAse H digestion of the RNA component.
Add “linkers” to the ends of the cDNA and ligate to a vector. |
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Term
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Definition
Isolate poly A+ mRNA.
Anneal a primer to the mRNA (often an oligo dT primer).
Make the first cDNA strand using mRNA as the template, a primer which is bound to the mRNA, dNTPs, and reverse transcriptase.
Synthesize the second cDNA strand using DNA polymerase.
RNAse H digestion of the RNA component.
Add “linkers” to the ends of the cDNA and ligate to a vector. |
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Term
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Definition
| autonomous replication; transformed into bacteria; have antibiotic selection; rarely used for genomic libraries in eukaryotes; inserts must be less than 10 kb; stored as a bacterial colony |
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Term
|
Definition
| autonomous replication; infected into bacteria; no antibiotic selection; used for genomic libraries; inserts between 12-20 kb; stored as phage lysate |
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Term
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Definition
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Term
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Definition
| re-association of DNA strands |
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Term
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Definition
COT describes the kinetics of hybridization between two nucleic strands in solution and is defined by the product of [nucleic acid] x (time). Put simply, when the concentration of two complementary strands in a solution is high, then it takes a shorter time for hybridization to occur than it does when one or both of the strands are present at a low concentration.
Cot curves plot percent reassociation versus Cot (mole secs/liter), and are used to measure the sequence complexity of DNA samples . DNA from organisms with small genomes have low sequence complexity and reassociate at much lower Cot values, than do denatured DNA samples from more complex organisms. |
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Term
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Definition
| Co(tl/2)=1/k where Co is the concentration of the single stranded DNA at t=0. |
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Term
| Factors Cot1/2 depends on |
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Definition
1) amount of DNA in reaction (↑ DNA → ↑ Cot )
2) salt concentration (high salt favors renaturation)
3) repetitiveness of DNA sequences (↑ DNA → ↓Cot) |
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Term
| Fast, Intermediate, and Slow regions of Eukaryotic Cot curve |
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Definition
Fast: 25% of the genome,340 bp complexity, repeated 500,000 times
Intermediate: 30 % of genome, 6x105 bp complexity, repeated 350 times
Slow: 45% of the genome, 3x108 bp complexity, repeated 1 time |
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Term
| Influence of salt and temp on stringency |
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Definition
| Stringency increases as salt decreases or temp increases |
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Term
| Purpose of Restriction fragment length polymorphism (RFLP) |
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Definition
| To follow a particular allele through a pedigree |
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Term
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Definition
| To obtain overlapping genomic clones such that an entire gene sequence can be obtained |
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Term
| Purpose of chromosome walking |
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Definition
| A method allowing one to obtain overlapping genomic clones |
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Term
| Purpose of genomic mapping |
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Definition
| To determine the positions within a gene at which restriction sites are present |
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Term
| Southern, Northern, and Western blots |
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Definition
Northern blots examine
RNA expression and transcript
length through DNA-RNA
hybridization. The full mRNA transcript length is detected. Southern blots examine DNA presence and size. Both of these procedures use nucleic
acid probes. Western blots
examine protein expression and
protein size through protein-antibody interactions; the antibody is the probe
in this procedure. |
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Term
|
Definition
Polyacrylamide gels are often used to separate proteins via electrophoresis.
SDS is often added beforehand to confer a large negative charge to the proteins,
thus allowing the separation to be based off size (molecular weight) of the proteins, rather than charge. This procedure is called SDS-PAGE for SDS-polyacrylamide gel electrophoresis. Note that the proteins migrate toward the (+) end and that smaller proteins move faster through the gel than larger proteins. The proteins can be visualized by staining the gel with Coomassie blue stain or silver stain following the electrophoresis.
The proteins can be transferred to nitrocellulose paper for Western blot analysis |
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Term
| How to use electrophoresis to determine pI |
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Definition
| By subjecting the protein mixture to an electric field of ampholytes, the proteins will migrate until their pI = the pH, where the net charge of the protein is 0. |
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Term
| types of small RNA molecules that do gene silencing |
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Definition
| siRNA (short interfering), shRNA (small hairpin), miRNA (micro). Each works through a different mechanism. |
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Term
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Definition
| cells are transfected with preformed siRNA duplexes, generally in combination with a transfection reagent. The transfected siRNA becomes incorporated into the RISC following cell entry, leading to degradation of target gene expression. |
|
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Term
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Definition
| transfected plasmid vectors must reach the nucleus for transcription of shRNA. The shRNA is processed and exported to thecytoplasm where it is incorporated into the RISC for directed gene silencing |
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Term
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Definition
| viral vectors encoding siRNA or shRNA bind to the target cell via a receptor, followed by receptor-mediated endocytosis |
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Term
| Duchenne's Muscular Dystrophy (DMD) |
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Definition
| X-linked recessive and caused by deletions in the dystrophin gene |
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Term
| How do cDNAs and genomic DNA correspond to each other? |
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Definition
| All of the cDNA sequence should map to genomic DNA fragments; however, only some of the genomic DNA will hybridize to cDNA sequence because of the non-hybridization that occurs with intron and intergenic sequences |
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Term
| How do genomic DNAs and RNAs correspond? |
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Definition
| Exons are mapped from genomic DNA by taking the genomic DNA fragments and hybridizing them to Northern blots; if a transcript is detected, then the genomic DNA has an exon. |
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Term
| Why is poly A+ RNA sometimes used on Northern blots? |
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Definition
| It is done to increase the sensitivity of detecting mRNA |
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Term
| How to isolate poly A+ RNA |
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Definition
| Pass total RNA through an oligo dT column. poly A mRNA will bind, but ribosomal and tRNA will not. mRNA can then be eluted with water. |
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Term
| In what tissues are full length DMD transcripts found? |
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Definition
| skeletal, cardiac and smooth muscle |
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Term
| Process of DNA sequencing using dideoxynucleotides |
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Definition
Clone the foreign DNA into the vector.
Anneal a primer 5’ of the cloned foreign DNA.Set up 4 tubes with the ddNTPs,
dNTPs, DNA polymerase (Klenow
fragment), and vector/foreign DNA.
Allow reaction to occur for 40 min
at 37 degrees centigrade.
Run acrylamide gel and read DNA
sequence in 5’ to 3’ direction (smallest
fragments are at the bottom). |
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Term
| How to determine if people have cystic fibrosis or are carriers? |
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Definition
| See if patient DNA hybridizes to allele-specific oligonucleotide probes (both normal and mutant) |
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Term
| Purpose of DNA microarray |
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Definition
| To examine the expression of thousands of genes in a single assay and to quantitatively determine the relative gene expression profile between two different populations of cells or tissues |
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Term
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Definition
| Spot 1000’s of DNA gene identifying sequences onto a matrix(i.e. glass slide). Competitively hybridize fluorescently-labeled cDNAs from the different cell populations, assay the fluorescent readout, and analyze with the computer. |
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Term
| Two common fluorophores used in DNA microarray |
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Definition
| Rhodamine (red) and Fluroscein (green) |
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Term
| In a microarray, what does green mean? |
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Definition
| represents Control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA. |
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Term
| In a microarray, what does red mean? |
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Definition
| represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA. |
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Term
| In a microarray, what does yellow mean? |
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Definition
| represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA (both expressed at equivalent levels). |
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Term
| In a microarray, what does black mean? |
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Definition
| represents areas where neither the Control nor Sample DNA hybridized to the target DNA. |
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Term
| What is important about color intensity in microarray? |
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Definition
| provides an estimate of the expression level of the gene(s) in the sample and control DNA. |
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Term
| Purpose of Polymerase Chain Reaction (PCR) |
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Definition
| used to amplify a specific DNA fragment millions of times for research or diagnostic purposes. With modifications, it can also be used to amplify RNA molecules. If desired, these amplifications can be performed in a quantified manner. |
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Term
| Essential requirements for PCR |
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Definition
1. Synthetic oligonucleotide primers (~20 nucleotides each) that are complementary to regions on opposite strands that flank the target DNA sequence and are oriented with their 3' hydroxyl ends towards each other.
2. A target sequence in a DNA (or RNA) sample that lies between the pair of primers which can be from 100 ‑ 5,000 nucleotides in length.
3. A thermostable DNA polymerase (usually Taq polymerase is used) that can withstand heating to 95oC or higher;
4. The four deoxyribonucleotides. |
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Term
|
Definition
PCR uses approx 30 cycles. Steps of each cycle:
1) Denaturation. Raising the temperature in the reaction tube to ~95oC causes the strands of the source DNA to denature into single strands. This reaction last ~1 min.
2) Renaturation. The temperature is slowly cooled (~2‑3 min) to 55oC allow the primers to base pair with their complementary sequences in the source DNA.
3) Synthesis. The temperature is raised to ~75oC so Taq DNA polymerase can function. DNA synthesis is initiated at the 3’‑hydroxyl end of each primer. This cycle lasts ~2 min. |
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Term
| How PCR can be used to determine if deletions of exons or alternative splicing occurs with a primary transcript? |
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Definition
| By using 2 primers corresponding to non-adjacent exons and amplifying the RNA, one can examine the products to determine if exons are missing or multiple mRNAs are produced. |
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