278
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
unchanged after amplification. Unfortunately, library amplification often results in the loss of clones which grow poorly. Amplifying plasmid
libraries in a semi-solid suspension culture, such as in a low-melting temperature agarose mixed with culture broth, minimizes this problem.
• Linkersthat ligate to the ends of cDNA molecules must be cleaved with a
restriction enzyme that generates cohesive ends prior to ligation to a similarly-prepared vector. Protocols that rely on linkers to enhance cloning efficiency must include a step that protects the restriction sites within
the cDNA from cleavage. This is accomplished in one of four ways. In
older protocols, the double-stranded cDNA was treated with a methylase
specific for the restriction enzyme sites prior to the ligation of linkers.
The linkers were then Iigated to the methylated cDNA, digested with the
appropriate restriction enzyme, and finally Iigated to a compatible vector. Incomplete methylation, however, resulted in restriction of internal
cDNA sites and produced clonesthat were of incomplete length. Analternative method uses linkers that contain restriction sites such as Noti
that are rarely found in cDN As. A significant advantage of this approach
is that methylation, which is often an inefficient process, is no Ionger
required. An additional benefit is a reduction in the number of steps
required to synthesize a library. A drawback is that some cDNAs do include rare restriction sites within their sequences (for example, there are
at least three genes within the polycystic kidney disease 1 (PKD 1) region
that have Noti sequences within their cDNAs). A third approach is to add
5-methyl-dCTP to the reaction mix for the synthesis of either the first or
both strands (Figure 1A). The hemi-methylated DNA will resist restriction with many enzymes. A newer approach that has even fewer steps
involves the use of Iinker-adapters. These synthetic oligonucleotides
have been designed with one end blunt, suitable for ligation to the
cDNA molecules, and one end that contains an overhang suitable for
ligation to a compatible vector.
• Oligod(T)-primed libraries often lack 5' end sequences. When primed by
oligo(dT), reverse transcriptase begins cDNA synthesis at the poly-A tail
and may "fall off" the molecule before copying its full length. Second
strand synthesis can also be an incomplete process. These problems
can be circumvented by constructing a library in which reverse transcription is primed using random primers, mixtures of short oligonucleotides
that have, on average, multiple annealing sites distributed along an
mRNA molecule. Inserts constructed using random primers, however,
are unlikely tobe full-length, and average-insert size tends tobe shorter
than in oligo-d(T) primed libraries.
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
unchanged after amplification. Unfortunately, library amplification often results in the loss of clones which grow poorly. Amplifying plasmid
libraries in a semi-solid suspension culture, such as in a low-melting temperature agarose mixed with culture broth, minimizes this problem.
• Linkersthat ligate to the ends of cDNA molecules must be cleaved with a
restriction enzyme that generates cohesive ends prior to ligation to a similarly-prepared vector. Protocols that rely on linkers to enhance cloning efficiency must include a step that protects the restriction sites within
the cDNA from cleavage. This is accomplished in one of four ways. In
older protocols, the double-stranded cDNA was treated with a methylase
specific for the restriction enzyme sites prior to the ligation of linkers.
The linkers were then Iigated to the methylated cDNA, digested with the
appropriate restriction enzyme, and finally Iigated to a compatible vector. Incomplete methylation, however, resulted in restriction of internal
cDNA sites and produced clonesthat were of incomplete length. Analternative method uses linkers that contain restriction sites such as Noti
that are rarely found in cDN As. A significant advantage of this approach
is that methylation, which is often an inefficient process, is no Ionger
required. An additional benefit is a reduction in the number of steps
required to synthesize a library. A drawback is that some cDNAs do include rare restriction sites within their sequences (for example, there are
at least three genes within the polycystic kidney disease 1 (PKD 1) region
that have Noti sequences within their cDNAs). A third approach is to add
5-methyl-dCTP to the reaction mix for the synthesis of either the first or
both strands (Figure 1A). The hemi-methylated DNA will resist restriction with many enzymes. A newer approach that has even fewer steps
involves the use of Iinker-adapters. These synthetic oligonucleotides
have been designed with one end blunt, suitable for ligation to the
cDNA molecules, and one end that contains an overhang suitable for
ligation to a compatible vector.
• Oligod(T)-primed libraries often lack 5' end sequences. When primed by
oligo(dT), reverse transcriptase begins cDNA synthesis at the poly-A tail
and may "fall off" the molecule before copying its full length. Second
strand synthesis can also be an incomplete process. These problems
can be circumvented by constructing a library in which reverse transcription is primed using random primers, mixtures of short oligonucleotides
that have, on average, multiple annealing sites distributed along an
mRNA molecule. Inserts constructed using random primers, however,
are unlikely tobe full-length, and average-insert size tends tobe shorter
than in oligo-d(T) primed libraries.