Term
| Asexual reproduction through artificial means is also known as... |
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Definition
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Term
| What are restriction endonucleases and where do they cut? |
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Definition
-enzymes in bacteria that cleave foreign DNA
-cut in the middle of a DNA segment ('endo') |
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Term
| These enzymes restrict DNA from replicating... |
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Definition
| restriction endonucleases |
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Term
True or False
Restriction endonucleases are more likely to cut a palindromic sequences than other sequecnes |
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Definition
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Term
| How many restriction sites can a single restriction endonuclease recognize? |
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Definition
| one specific sequence (restriction site) only |
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Term
| What are the two types of restriction sites? |
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Definition
| cohesive ends & blunt ends |
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Term
| What causes 'staggered' ends of restriction fragments? |
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Definition
| -REs cut at different nucleotide positions on the two DNA strands |
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Term
| What causes restriction fragments to have blunt ends? |
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Definition
| -REs cut at the same position on the two DNA strands |
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Term
| What is a cloning vector? |
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Definition
| autonomous replicating DNA used to amplify & manipulate recombinant DNA |
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Term
| What are the essential components of a cloning vector? |
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Definition
-replication origin
-selectable marker
-one or more unique RE sites |
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Term
| Why is it important for RE sites of a cloning vector to be unique? |
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Definition
| so the bacteria DNA is only cut once |
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Term
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Definition
Multiple Cloning Site
-string of unique restriction sites on a cloning vector |
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Term
| Where do plasmid vectors mostly originate from? |
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Definition
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Term
| Why is it important for plasmid vectors to have a maximum insert size of 15kbp? |
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Definition
| any more than this and replication success will be poor, you might lose the piece of DNA you inserted |
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Term
| What are the two types of special purpose vectors? |
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Definition
Expression Vectors
Shuttle Vectors |
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Term
| What is the purpose of expression vectors? |
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Definition
| expressing protein in cells |
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Term
| What is the purpose of shuttle vectors? |
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Definition
| -have different replication origins, can replicate in both E.coli and another species |
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Term
| What are the steps of cloning DNA in a plasmid vector? |
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Definition
DALTS
1. Digestion
2. Annealing
3. Ligation
4. Transformation
5. Selection |
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Term
| What is the special characteristic of the polymerase used in PCR? |
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Definition
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Term
| What are the steps of a PCR cycle? |
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Definition
1. Denaturation
2. Annealing
3. Extension |
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Term
True or False
PCR is a sensitive process |
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Definition
| True - can isolate & amplify specific DNA from very little template DNA from a mixed source |
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Term
| What is the disadvantage of Taq polymerase in PCR? |
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Definition
| -doesn't have proofreading capabilities, so nucleotide substitutions might occur & be amplified |
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Term
| What is gel electrophoresis? What are its applications? |
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Definition
separation of a mix of DNA fragments
-check size of DNA fragments
-isolate DNA fragments for cloning |
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Term
| Do larger fragments move faster or slower in gel electrophoresis? |
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Definition
| slower (speed is inversely proportional to size) |
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Term
| DNA's have a ______ charge and migrate to the anode |
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Definition
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Term
| What are the two steps of Southern blotting? |
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Definition
1. Transfer & immobilize DNA from electrophoretic gels onto membrane
-Detect DNA fragments through molecular hybridization |
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Term
| What is molecular hybridization? |
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Definition
| annealing a DNA/RNA strand to its complement |
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Term
| What are the applications of Southern Blotting? |
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Definition
-detect presence of a DNA fragment
-detect homologous DNA
-genetic mapping (RFLP) |
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Term
| What are some techniques for analyzing RNA? |
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Definition
- RNA electrophoresis
- Northern Blotting
- RT-PCR |
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Term
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Definition
| Reverse Transcriptase Polymerase Chain Reaction |
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Term
True or False
Northern Blotting requires restriction digestion |
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Definition
| False, only Southern Blotting does |
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Term
| Why aren't RE's required for Northern Blotting? |
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Definition
| RNA is already found in small fragments, so doesn't need to be cut |
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Term
| What are the two steps of RT-PCR? |
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Definition
1. reverse transcription of single-stranded RNA to double-stranded cDNA
2. PCR amplification of cDNA |
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Term
| What is an application of RT-PCR? |
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Definition
| determining the transcription levels of a gene |
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Term
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Definition
| determining the nucleotide sequence of a segment of DNA |
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Term
| What are the components required for dideoxy nucleotide sequencing? |
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Definition
template DNA
sequencing primer
DNA polymerase
dNTPs |
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Term
| What would be the best method of identifying functional domains in a protein, or coding sequences of a gene? |
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Definition
| Sanger's Dideoxy Nucleotide Sequencing |
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