Common types of DNA Libraries:

cDNA: prepared from mRNA via reverse transcriptase (worry that all genes are not represented or that they are not full length, i.e. split genes)

genomic library: chromosomal DNA usually carried as DNA inserts in bacteriophage lambda

expression library: employed for cloning by complementation. DNA must be in an expression vector.

General outline for preparation of a library:

a. isolate DNA by cleaving with restriction endonucleases and separating the DNA fragments by gel electrophoresis in agarose.

b. make recombinant DNA molecules by combining the DNA fragments from step "a" with vector DNA (cleaved with same restriction endonucleases).

c. add DNA ligase and ATP to covalently join the insert DNA to the plasmid DNA.

Cloning DNA

a. probe the library to find the copy of the gene of interest. Use knowledge of the sequence of conserved areas of the protein to make oligonucleotide, use antibodies to your protein (requires expression vector), or use gene complementation.

b. isolate the colony containing the DNA insert of interest (the clone)

c. amplify the amount of DNA (of this gene) you have by growing E. coli with plasmid or infected with a lambda phage clone.

General Considerations in Sequencing DNA

To sequence DNA you must be able to:

1. isolate a segment of DNA of unique sequence.

2. sequence a single strand.

3. label the portions being sequenced (with radioactivity or dyes)

4. produce breaks in the synthesized DNA (or sample DNA) that occur at each of the four different bases (A, T, G, C) and at every site in the sequence where this base occurs.

5. separate the fragments that are produced using a method that resolves DNA fragments that differ by one nucleotide in size.

6. identify each fragment and associate it with the identity of its 3' terminal nucleotide.

The Sanger Method (dideoxy method)

1. uses M13 bacteriophage, which consists of ss DNA.

2. isolate ds replicative form --> do restriction digest ---> insert fragment of DNA to be sequenced (DNA ligase).

3. grow M13 (with your DNA) in E. coli to obtain ss DNA.

4. hybridize an oligo primer (universal) to M13 DNA.

5. extend from this primer using 4 incubations; each containing one of the four dNTP's as the dideoxy compound (1/10 ratio of dideoxy to deoxy) + other four dNTP's (one or more radioactive).

6. add DNA polymerase and allow reaction to proceed for fixed time.

7. separate oligonucleotide fragments by acrylamide gel electrophoresis, expose x-ray film to gel, and read the sequence.

General considerations:
1. must be able to add residues via chemical reaction one at a time.

2. reaction must go far toward completion to avoid mixed sequences [(0.98)11 = 0.80, but (0.90)11 = 0.3].

3. must avoid side reactions by blocking other reactive sites.

4. attach to solid phase support to facilitate "purifications" between steps.

Chemical reactions:
a. remove DMTr (exposes 5' OH for reaction)
b. add next residue by adding activated form (3' phosphoramidites)
c. oxidize phosphoramidite --> methyl phosphate
d. return to step "a."
e. when required sequence has been synthesized --> go to f.

To release unmodified DNA:
f. remove blocking groups on bases
g. remove methyl groups from phosphates
h. cleave from silica

Sequencing DNA: Maxam/Gilbert method
Chemistry that permits cleavage of DNA at the site of particular bases in a DNA sequence:

the purine lanes (A and G) done via depurination (depurination will occur at pH = 2 at 4C overnight)

a. methylation (via dimethylsulfate) of guanine at N-7 (faster reaction) and adenine at N-3 makes depurination easier.

b. depurination by heating at pH 7

c. break the backbone and eliminate the sugar molecule by heating in alkali (breaks at guanine --> more quickly when methylated, yields the "G" lane).

c'. methylated adenine is less stable than Me-G in dilute acid. Gives preferential release of A (but get both),yields the "A + G" lane

the pyrimidine lanes (C and T)
a. cytosine and thymine split by hydrazine = H2N-NH2 (breaks down the ring).

b. backbone cleaved by piperidine (saturated pyridine), yields the "C + T" lane.

c. the reaction above is suppressed at T's by 2M NaCl, yields a "C" alone lane.