Term
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Definition
| Used to cut DNA, ESP for cloning experiments. Bind to specific base sequence and cleave DNA backbone at two drives locations one on each strand. Made naturally by bacteria I protect from invasion by foreign DNA. Can digest DNA into fragments with sticky ends. Example - EcoRI. Usually recognize palindromic sequences. (Identical when read in the opposite room in complementary strand : 5'GAATTC'3 |
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Term
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Definition
| Created when restriction endonucleases cut DNA fragments. Single stranded regions of DNA that can hydrogen bond to complementary sequence from diff DNA source. Then DNA ligase catalyze a covalent bond formation in sugar-phosphate backbones of the strands. |
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Term
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Definition
1. Cut DNA of plasmid and of chromosomal DNA from human cell with same restriction enzyme. (Restriction digest)
2. Ligation of fragments via DNA ligase
3. Mix vectors with ecoli
4.transformstion (living cells take up extra cellular DNA)
5. Grow ecoli on selective agar plate so transformed cells grow (ampR) and are white (xgal) via lacz gene for recombinant plasmid selection.
6. Each bacterial colony derived from single bacterium cell, so all cells have same plasmid DNA. |
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Term
| cDNA can be made from mRNA via reverse transcriptase. |
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Definition
1. mRNA mixed with a poly-dT primer which is complementary to the 3' poly-A tail of mRNA.
2. Add reverse transcriptase and dNTPs (deoxyribonucleotides) to make DNA strand complementary to mRNA.
3. Add RNaseH, DNA polymerase, DNA ligase.
4. RNaseH partially digests RNA creating little fragments that act as RNA primers, used by DNA pol to make complementary DNA strand. DNA ligase seals the deal.
5. When DNA is made from RNA, DNA is called complementary na (cDNA).
Advantages:
Lacks introns (large) so easier to insert into vectors. |
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Term
| How to figure out where restriction sites are |
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Definition
| Via DNA sequencing or restriction mapping |
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Term
| Steps in restriction mapping |
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Definition
1. Plasmid isolated and placed in separate test tubes containing a particular restriction enzyme or combo thereof
2.after digestion, fragments are separated by gel electrophoresis
3. To determine sizes of fragments in the lanes, try are compared to lane 1 ladder with known markers. |
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Term
| PCR (polymerase chain reaction) steps |
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Definition
Overview: region of DNA flanked by complementary primers is amplified. each cycle involves denaturation, primer annealing, and primer extension.
reagents for pcr: sample of DNA with gene of interest (template DNA), 2 primers on either side of gene, dNTPs, DNA polymerase that can withstand heat (ie Taq polymerase)
Steps:
1. Denature template DNA by heat (95deg), cause strands to separate.
2. Lower temp (50-60 deg) and primers anneal to DNA
3. Raise temp slightly (72 deg) and taq pol catalyzes synthesis of complementary strand of DNA starting at primers (primer extension) doubling the amt of DNA.
4. Repeat cycle many times in chain rxn. 4 cycles yields 2^4 or 16 copies of region of interest. |
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Term
| Reverse transcriptase PCR (RT-PCR) use |
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Definition
| Can be used to so lift RNA. Detects and quantifies amt of specific RNAs in living cells. Requires RNA, dntps, Reese transcriptase, and a primer binding near 3' end of gene of interest. Generate single area des cDNA, template DNA in conventional PCR rxn. RNA is amplified to produce many compiles of DNA. |
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Term
| Real-time PCR (qPCR sometimes called RT-PCR) |
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Definition
Allows quantitation of products in real time and you can determine how much DNA OR mRNA was originally in the sample before PCR.
The thermocycler can measure changes in level of fluorescence emitted by detector molecules added to PCR mixture.
One detector molecule is an oligonucleotode called TaqMan. As pcr products accumulate, more and more of the detectors are digested, therefore level of fluorescence increases. Measure during exponential phase (early) |
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