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51 Cards in this Set
- Front
- Back
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What is the purpose of molecular cloning? |
It is to isolate a particular gene or other DNA sequence in large quantities for further study. |
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What is the method molecular cloning? |
*transfer of a DNA sequence of interest into a single cell of a microorganism *the microorganism is subsequently grown and reproduces the DNA sequence along with its own DNA |
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How is a clone defined? |
Every individual microorganism in the colony is derived from that original single cell and contains the same identical transferred segment of DNA. |
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How is molecular cloning defined? |
The entire process of growing large quantities of the sequence of interest. |
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What is needed for detailed molecular analysis? |
Large quantities of the sequence of interest can be isolated in pure form from an individual clone for detailed analysis. |
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What is DNA recombination? |
The ligation of DNA molecules from different sources
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What is a vector? |
A DNA molecule that can replicate autonomously (by itself) in a host cell. |
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How many copies can a vector make? |
It can often achieve a HIGH number of copies per cell. |
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What are plasmids? |
*circular double stranded DNA *can be replicated independently *carry antibiotic resistance |
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What three components make up a plasmid? |
*an origin of replication *one or more selectable markers *one or more restriction sites |
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What does endonuclease do? |
It cleaves DNA to be cloned and a plasmid--these are then ligated. |
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What does a chimeric plasmid contain? |
Plasmid DNA and foreign DNA |
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To obtain DNA during DNA cloning, what must be done? |
*Isolate plasmids
*cleave with restriction endonuclease *isolate cloned DNA |
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To obtain protein during DNA cloning, what must be done? |
*grow under conditions that allow expression of a cloned gene *isolate protein |
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What do restriction enzymes recognize? |
They recognize specific double stranded sequences that are palindromes. |
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What do restriction enzymes cleave? |
They cleave phospho-diester backbones at or near the recognition site. |
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What results from the cleaving of phosphodiester backbones? |
*sticky ends *blunt ends |
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What is a DNA sequence palindrome? |
Each strand of the DNA, when read in the 5' to 3' direction, has the same sequence |
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How is recombinant DNA molecules made? What kind of enzymes are used? |
Sticky ends are connected, the fragments base-pair and are joined by DNA ligase to make recombinant DNA. Restriction enzymes and DNA ligase is used. |
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What happens to target DNA in recombinant DNA? |
Target DNA is inserted with a restriction enzyme *same enzyme is used in vector DNA |
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What happens to vector DNA in recombinant DNA? |
Vector DNA are cleaved with a restriction enzyme. *same enzyme is used in target DNA |
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What does an autoradiogram do? |
It can be used to identify bacterial colonies on the original agar plate that contains the desired DNA sequence. |
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What are DNA libraries? |
They are collections of restriction fragments cloned within suitable host cells. |
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What is a genomic DNA library? |
*each clone contains a fragment of the entire genome of an organism
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How are genomic DNA libraries generated? |
They are generated by deliberately using limiting amounts of a restriction enzyme that cuts at sites present at high frequency in the genome. |
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What does a genomic DNA library contain? |
It theoretically contains all of the nuclear DNA sequences. (includes RNA types found in the nucleus and could make ribosomes from this) |
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What is cDNA? |
Has all the normal components except the introns because it is made by reverse transcriptase of mRNA. |
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What is contained in the cDNA library? |
It contains hybrid vectors with cDNA inserts. Each clone contains an intact gene. Each clone has NO introns. |
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What are screening libraries used for? |
They are used to identify the clone carrying the DNA insert of interest. It is done by nucleic acid hybridization. |
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What charge does DNA have? |
negative charge |
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In electrophoresis, which direction does the DNA go or migrate to? |
In the positive direction. |
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Which side has larger molecules and smaller molecules after electrophoresis? |
*larger molecules are near the negative end *smaller molecules are near the positive end |
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After electrophoresis, what ends of the DNA are where? (5' and 3') |
*The 5' end is on the positive end. *The 3' end is on the negative end. |
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What is a Southern Blot used for? |
It is used to identify DNA sequences on gel. |
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When is a Southern Blot produced? |
It is produced when DNA on a nitrocellulose blot of an electrophoretic gel is hybridized with a DNA probe. |
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What does a Northern Blot identify? |
It identifies RNA sequences on a gel. |
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When is a Northern Blot produced? |
It is produced when RNA on a nitrocellulose blot is hybridized with a DNA probe. |
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What does a Western Blot identify? |
It identifies a protein molecule on a gel. |
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What is involved in making a Western Blot? |
It involves separating proteins b gel electrophoresis and probing with labeled antibodies for specific proteins. |
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Describe Southern Blotting. |
*prepare genomic DNA from lymphocytes obtained by routine venipuncture (blood draw) *cleave by restriction enzymes *separate by agarose gel electrophoresis *denatures with a strong base *transferred from the gel to a filter paper *hybridization using single stranded labeled probe |
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Where is Southern Blotting used? |
On crime scenes and hospitals. Can be used to test for sickle cell anemia. You compare your prepared DNA with a control. |
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What is polymerase chain reaction used to make? |
It is used to make many copies of target DNA sequence. It is necessary to have enough starting template for sequencing. |
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What materials are needed for PCR? |
*DNA template *Deoxyribonucleotide bases (dATP, dTTP, dCTP, dGTP) *Thermostable DNA polymerase *PCR buffer *Magnesium |
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What does PCR buffer do? |
It makes optimal conditions for enzyme activity |
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Why is magnesium needed for PCR? |
*too few: lower enzyme activity *too much: decrease fidelity (more errors) |
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What are the 3 steps of PCR? |
*denaturation *annealing *extension *repeated for 20 to 40 cycles |
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What happens during the denaturation step of PCR? |
Heat at 94 to 96C to separate the double strands (hydrogen bonds) *the double strand melts open to single stranded DNA, all enzymatic reactions stop (including extension from a previous cycle) |
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What happens during the annealing step of PCR? |
*Primers hybridize with the template DNA *temp is 50 to 70C *H bonds are constantly formed and broken between the single stranded primer and the single stranded template |
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What happens during the extension step of PCR? |
*polymerase adds nucleotides to the hybridized primers *68 to 72C (optimal temp of Taq polymerase--came from an enzyme from a geyser) |
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During the extension phase of PCR, where do the bases couple? How does it read? |
The bases are coupled to the primer on the 3' side by polymerase. The polymerase adds from 5' to 3' and it reads the template from 3' to 5'. The bases added are complementary to the template. |
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After PCR, what is done with the DNA? |
You run it through a gel to make sure that it worked well. |
