Methods for Working with DNA and RNA

4. DNA sequencingA. Sanger dideoxy method

Use chain terminators and DNA polymeraseEnzymatic approach

B. Maxam-Gilbert method Chemical approach: nucleotide-specific modification

Methods for Working with DNA and RNA

Sanger dideoxy methodi. use of dideoxynucleotides as chain terminatorswill have four separate reactions for each chain

terminatorii. Klenow fragment of DNA polymeraseiii. single-stranded DNA template annealed to an

oligonucleotide primer iv. alpha P-32 dATP typically used to internally

labeled synthesized DNA figure 7-15

Automated DNA sequencing

Fluorescent tagged DNA

Methods for Working with DNA and RNA

5. DNase I footprinting and Interference AssayA. radiolabel one unique 5' end of the DNA using T4 polynucleotide kinase and alpha-P32 ATP.B. after binding protein to DNA, add in DNaseI(an endonuclease) to cut the regions of DNA not bound by proteinC. It is key to cut the DNA only once per DNA molecule

Methods for Working with DNA and RNA

6. cDNA– an oligo dT primer is annealed to the poly A

portion of the mRNA template– the enzyme reverse transcriptase catalyzes the

synthesis of DNA from an RNA template– reverse transcriptase backs around to place a

"hook" at the end - DNA-DNA primer– RNA is removed by alkaline hydrolysis – DNA synthesis is completed with DNA

polymerase– This process is used in molecular cloning see

next section

Methods for Working with DNA and RNA

7. PCR or polymerase chain reaction A. An effective method for in vitro amplification of a particular DNA sequencing

as much as a million times amplification with 25 cyclesB. relies on using a thermostable version of DNA polymerase

example is Taq DNA polymeraseC. can be used for

molecular cloning (also RT-PCR, combine PCR with synthesis of cDNA)

site-directed mutagenesisrapid identification of clonerapid DNA sequencing to name just a few

D. Extreme care needs to be taken to prevent contaminationeven dust can be significant

Chemical Synthesis of DNA

Solid-phase chemistry basedSynthesis of oligonucleotide

DMT = dimethoxytritylRemoved by acid treatment

Phosphoramidite Method

Questions

• Why are protein expression vectors useful? • What kind of cells are proteins generally over-

expressed in and why? • What are the considerations in vector construction

that need to be taken into account with over-expression of a protein?

• Why are cDNA libraries still so important in the era of genomics?

• What information can be derived from cDNA that are not obtained generally by DNA microarrays?