3.4 DNA Library
For decades, the term “DNA library” was used to refer to a collection of DNA
sequences from an organism of interest that had been ligated into a vector (e.g., a
plasmid) and used to transform bacterial cells. The DNA library was the collection of
bacterial cells containing the recombinant (vector/insert) molecules. Eventually the
term was also used to describe a collection of virions each containing a genome into
which a piece of foreign DNA had been ligated. Regardless if the foreign DNA
inserts were pieces of nuclear DNA, the library was deemed a genomic library. If the
inserts were reverse-transcribed mRNA molecules (i.e., cDNAs), the term cDNA
library was used to describe it. If the vector contained elements that encouraged
transcription and translation of the inserts, the term expression library was utilized.
Unfortunately, when so-called next-generation sequencing (NGS) came along,
the term DNA library was co-opted to simply mean DNA prepared for sequencing
on a NGS instrument. Preparation of DNA for NGS is platform dependent, although
typically genomic DNA or DNA prepared from reverse-transcribed RNA molecules
is sheared into fragments of a particular length (by sonication, nebulization, etc.) and
quantified. For many NGS instruments, the remaining preparation process often
involves ligation of adapters onto the ends of DNA fragments.
3.5 Physical Mapping
The term “physical mapping” can be used to describe the identification of relatively
large pieces of partially overlapping DNA collectively spanning a chromosome or
part of a chromosome and/or determination of nucleotide distances between genes
and markers. There are numerous physical mapping techniques that can be utilized
separately or together. Some of these techniques rely upon the existence of largeinsert genomic libraries, most notably bacterial artificial chromosome (BAC) libraries. Each BAC clone can carry a relatively large piece of genomic DNA (>100 kb)
(see Peterson et al. 2000 for review). A few of the physical mapping techniques that
are utilized include:
1. DNA fingerprinting – BACs can be digested with a series of restriction enzymes
and separated by gel electrophoresis. The resulting banding patterns, known as
fingerprints, can be compared as a way to identify partially overlapping BACs.
2. BAC-end sequencing (BES) – BAC inserts are cloned into a vector of known
sequence, and consequently primers complementary to the vector can be used to
sequence the regions at each end of a BAC (these sequences are called BAC-end
sequences or BESs, and they are a type of paired-end sequence – see below).
BESs mark the termini of a BAC insert whose size can be determined by pulsedfield gel electrophoresis; the distance between the two BESs can be elucidated.
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D. G. Peterson and M. Arick
For decades, the term “DNA library” was used to refer to a collection of DNA
sequences from an organism of interest that had been ligated into a vector (e.g., a
plasmid) and used to transform bacterial cells. The DNA library was the collection of
bacterial cells containing the recombinant (vector/insert) molecules. Eventually the
term was also used to describe a collection of virions each containing a genome into
which a piece of foreign DNA had been ligated. Regardless if the foreign DNA
inserts were pieces of nuclear DNA, the library was deemed a genomic library. If the
inserts were reverse-transcribed mRNA molecules (i.e., cDNAs), the term cDNA
library was used to describe it. If the vector contained elements that encouraged
transcription and translation of the inserts, the term expression library was utilized.
Unfortunately, when so-called next-generation sequencing (NGS) came along,
the term DNA library was co-opted to simply mean DNA prepared for sequencing
on a NGS instrument. Preparation of DNA for NGS is platform dependent, although
typically genomic DNA or DNA prepared from reverse-transcribed RNA molecules
is sheared into fragments of a particular length (by sonication, nebulization, etc.) and
quantified. For many NGS instruments, the remaining preparation process often
involves ligation of adapters onto the ends of DNA fragments.
3.5 Physical Mapping
The term “physical mapping” can be used to describe the identification of relatively
large pieces of partially overlapping DNA collectively spanning a chromosome or
part of a chromosome and/or determination of nucleotide distances between genes
and markers. There are numerous physical mapping techniques that can be utilized
separately or together. Some of these techniques rely upon the existence of largeinsert genomic libraries, most notably bacterial artificial chromosome (BAC) libraries. Each BAC clone can carry a relatively large piece of genomic DNA (>100 kb)
(see Peterson et al. 2000 for review). A few of the physical mapping techniques that
are utilized include:
1. DNA fingerprinting – BACs can be digested with a series of restriction enzymes
and separated by gel electrophoresis. The resulting banding patterns, known as
fingerprints, can be compared as a way to identify partially overlapping BACs.
2. BAC-end sequencing (BES) – BAC inserts are cloned into a vector of known
sequence, and consequently primers complementary to the vector can be used to
sequence the regions at each end of a BAC (these sequences are called BAC-end
sequences or BESs, and they are a type of paired-end sequence – see below).
BESs mark the termini of a BAC insert whose size can be determined by pulsedfield gel electrophoresis; the distance between the two BESs can be elucidated.
124
D. G. Peterson and M. Arick