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DNA sequencing: sanger method
- Prepare four tubes with the same DNA as the template, all four dNTP: A, T, G, C, DNA polymerase, and a DNA primer.
- Add concentration of a different ddNTP to each tube.
- Four sets of complememntary sequences are generated from template DNA.
- Separate the labled fragments by electrophoresis.
- Go down lanes and list the bases in the order they are fragmented.
- Use: for sequencing.
- Cloning vector is cleaved with restriction endonuclease.
- DNA of interest is obtained by cleaving chromosome with RE.
- Fragments are ligated to cloning vector.
- DNA is introduced to host cell.
- Propagation of transformed cell produces recombinant DNA.
- Function: make plasmid copies.
YAC and BAC:
- YAC: yeast artificial chromisome. just a bigger cloning vector.
- BAC: bacterial artificial chomosome. large plasmid construct.
- Purpose: to replicate specific DNA sequences many times.
- Heat to denature (separate strands).
- Bind oligonucleotide primers to the specific sequence (need to know sequence).
- Add heat stable DNA polymerase.
- Duplicate the DNA sequence between the primers.
- Repeat the above steps 20-30 times.
- Function: show is DNA sequence is in sample.
- (1) separate DNA segments by size in a gel (agarose gel)
- (2) transfer separated segments to membrane
- (3) identify segments. Cleave DNA molecule via restriction enzymes =>Agarose gel electrophoresis => fragments fractioned by size in gel => transfer to nitrocellulose filter (under denaturing conditions) => Nitrocellulose fiber with DNA fragments positioned identically to those in gel => hybridization with radioactively labeled DNA probe => X-ray film => radiograph showing hybrid DNA.
- Purpose: Identifies genesequences in a whole genome or plasmid.
- Allow DNA replication to begin at origin.
- Break DNA into fragments, flurecently lable DNA.
- Add labeled fragments into DNA microarray.
- Allow complememntary base pairing to occur.
- Examine microarray with flourencent scanner.
- Microarrays:Identifies gene sequences in a whole genome or plasmid. It is quantitative, youcan tell how the intensity light changes over time (logarithmic orexponential). This technique can be used on any biomolecule: lipids, carbs,etc.. Each spot contains multiple exact copies of the same sequence, each spothas a unique sequence, and probes hybridize in a complementary fashion.