recombinant DNA technology
a set of molecular techniques
for locating, isolating, altering, and studying DNA segments
genetic engineering
Genes
from two different bacteria might be joined, for example, or
a human gene might be inserted into a viral chromosome.
Commonly called genetic engineering
biotechnology
Recombinant DNA technology and other molecular
techniques are also being used to create a number of commercial
products, including drugs, hormones, enzymes, and
crops
restriction enzyme or restriction endonuclease
recognize and make
double-stranded cuts in DNA at specific nucleotide sequences
cohesive end
sticky ends, because
they are complementary to each other and can spontaneously pair to connect the fragments
gel electrophoresis
Electrophoresis is a standard biochemical technique for separating molecules on the basis of their size and electrical charge. A porous gel is often made from agarose (a polysaccharide isolated from seaweed), which is melted in a buffer solution and poure
autoradiography
a piece of X-ray film is
placed on top of the gel. Radioactively labeled DNA can be detected
probe
a DNA or RNA
molecule with a base sequence complementary to a sequence
in the gene of interest.
gene cloning
One way to amplify a specific
piece of DNA is to place the fragment in a bacterial cell and
allow the cell to replicate the DNA.
cloning vector
a stable, replicating DNA molecule
to which a foreign DNA fragment can be attached for introduction
into a cell.
cosmid
plasmids that are packaged into empty
viral protein coats and transferred to bacteria by viral infection.
bacterial artificial chromosome (BAC)
vectors originally constructed from the F plasmid - a special plasmid that controls mating and the transfer of genetic material in some bacteria; see Chapter 6) and can hold very large fragments of DNA that can be as long as 300,000 bp
expression vector
contains sequences required for transcription
and translation in bacterial cells
polymerase chain reaction (PCR)
allows DNA fragments to be amplified a billionfold within just a few hours. two essential requirements: 1) a single-stranded DNA template from which a new DNA strand can be copied and 2) a primer with a 3-OH group to which new nucleotides can be added
Taq polymerase
DNA polymerase was isolated
from the bacterium Thermus aquaticus, which lives in the
boiling springs of Yellowstone National Park. This enzyme,
dubbed Taq polymerase, is remarkably stable at high temperatures
DNA library
A collection of clones containing all the DNA fragments
from one source
genomic library
containing all the
DNA sequences found in the human genome
cDNA library
contains only DNA sequences that are
transcribed into mRNA; a cDNA library is created from
mRNA that is first converted into DNA and then cloned
into bacteria.
positional cloning
This approach�to isolate
genes on the basis of their position on a gene map
restriction fragment length polymorphism (RFLP)
variations
(polymorphisms) in the patterns of fragments produced
when DNA molecules are cut with the same
restriction enzyme
DNA sequencing
A powerful molecular method for analyzing DNA is a technique
dideoxyribonucleoside triphosphate
(ddNTP)
a special nucleotide is used as one of the
substrates
DNA fingerprinting
The use of DNA sequences to identify individual persons
microsatellite or short tandem repeat (STR)
very short
DNA sequences repeated in tandem and are found widely in
the human genome.
forward genetics
This approach, which begins with a phenotype (a
mutant individual) and proceeds to a gene that encodes the
phenotype,
reverse genetics
A geneticist might
begin with a gene of unknown function, induce mutations in
it, and then look to see what effect these mutations have on
the phenotype of the organism
transgene
An organism
that has been permanently altered by the addition of a DNA
sequence to its genome is said to be transgenic, and the foreign
DNA that it carries is called a transgene
knockout mice
A useful variant of the transgenic approach is to produce
mice in which a normal gene has been not just mutated, but
fully disabled
knock-in mice
Mice
that carry inserted sequences at specific locations
gene therapy
the direct transfer of genes into
humans to treat disease
genomics
the field of genetics that attempts to understand
the content, organization, function, and evolution of genetic
information contained in whole genomes
structural genomics
concerns the organization and
sequence of genetic information contained within a genome
genetic map
provide a
rough approximation of the locations of genes relative to the
locations of other known genes
physical map
are based on the direct analysis of DNA, and
they place genes in relation to distances measured in number of base pairs, kilobases, or megabases
map-based sequencing
short
sequenced fragments are assembled into a whole-genome
sequence by first creating detailed genetic and physical maps
of the genome, which provide known locations of genetic
markers (restriction sites, other genes, or known DNA
sequences) at regularly
contig
A set of two or more overlapping DNA fragments that
form a contiguous stretch of DNA
whole-genome shotgun sequencing
small-insert clones are
prepared directly from genomic DNA and sequenced
single-nucleotide polymorphism (SNP)
A site in the genome where individual members of a
species differ in a single base pair
haplotype
The specific set of SNPs and other genetic variants
observed on a single chromosome or part of a
chromosome
bioinformatics
an emerging field consisting of
molecular biology and computer science that centers on
developing databases, computer-search algorithms, geneprediction
software, and other analytical tools that are used
to make sense of DNA-, RNA-, and protein-sequence da
functional genomics
Functional genomics characterizes what the sequences do�
their function
transcriptome
The goals of functional genomics include the
identification of all the RNA molecules transcribed from a
genome,
proteome
all
the proteins encoded by the genome
homologous genes
Genes that are evolutionarily related are said to
be homologous
microarray
Microarrays rely on nucleic acid hybridization, in
which a known DNA fragment is used as a probe to find
complementary sequences
comparative genomics
the field of genomics that compares similarities
and differences in gene content, function, and organization
among genomes of different organisms
gene desert
In some areas of the genome, long stretches of DNA, often
consisting of hundreds of thousands to millions of base pairs
are completely devoid of any known genes or other functional
sequences; these regions are known as gene deserts
proteomics
the study of the proteome
mass spectrometry
a method for precisely determining the
molecular mass of a molecule
protein microarray
are similar to the microarrays used
for examining gene expression
Concept 1
Molecular genetics and recombinant DNA technology are used to locate, analyze, alter, study, and recombine DNA sequences. These techniques are used to probe the structure and function of genes, address questions in many areas of biology, create commercial
Concept 2
Molecular genetic analyses require special methods because individual
genes make up a tiny fraction of the cellular DNA and they
cannot be seen.
Concept Check 1
Briefly outline the steps required to genetically engineer bacteria
that will produce a protein encoded by a human gene.
First, the gene must be located and isolated from the rest of the genomic DNA. Then, the gene must be inserted into bacteria in a form that is stable and will be replicated. The gene must be placed in the bacteria in a way that ensures that it will be tra
Concept 3
Restriction enzymes cut DNA at specific base sequences that are palindromic. Some restriction enzymes make staggered cuts, producing DNA fragments with cohesive ends; others cut both strands straight across, producing blunt-ended fragments. There are fewe
Concept Check 2
Where do restriction enzymes come from?
Restriction enzymes exist naturally in bacteria, which use
them to prevent the entry of viral DNA.
Concept 4
DNA fragments can be separated, and their sizes can be determined
with the use of gel electrophoresis. The fragments can be
viewed by using a dye that is specific for nucleic acids or by labeling
the fragments with a radioactive or chemical tag.
Concept Check 3
DNA fragments that are 500 bp, 1000 bp, and 2000 bp in length
are separated by gel electrophoresis.Which fragment will migrate
farthest in the gel?
a. 2000-bp fragment c. 500-bp fragment
b. 1000-bp fragment d. All will migrate equal distan
c. 500-bp fragment
Concept 5
DNA fragments can be inserted into cloning vectors, stable pieces
of DNA that will replicate within a cell. A cloning vector must have
an origin of replication, one or more unique restriction sites, and
selectable markers. An expression vector contains se
Concept Check 4
How is a gene inserted into a plasmid cloning vector?
The gene and plasmid are cut with the same restriction
enzyme and mixed together. DNA ligase is used to seal nicks in
the sugar-phosphate bonds.
Concept 6
The polymerase chain reaction is an enzymatic in vitro (in a test
tube) method for rapidly amplifying DNA. In this process, DNA is
heated to separate the two strands, short primers attach to the
target DNA, and DNA polymerase synthesizes new DNA strands
f
Concept 7
One method of finding a gene is to create and screen a DNA
library. A genomic library is created by cutting genomic DNA into
overlapping fragments and cloning each fragment in a separate
bacterial cell. A cDNA library is created from mRNA that is converte
Concept 8
A DNA library can be screened for a specific gene by using complementary
probes that hybridize to the gene. Alternatively, clones in
a gene library can be examined for the protein product of the gene.
Concept 9
Restriction fragment length polymorphisms are variations in the
pattern of fragments produced by restriction enzymes, which
reveal variations in DNA sequences. They are used extensively in
gene mapping.
Concept 10
DNA can be rapidly sequenced by the dideoxy method, in which
ddNTPs are used to terminate DNA synthesis at specific bases.
Automated sequencing methods allow tens of thousands of base
pairs to be read in just a few hours.
Concept Check 5
In the dideoxy sequencing reaction, what terminates DNA synthesis
at a particular base?
a. The absence of a base on the ddNTP halts the DNA polymerase.
b. The ddNTP causes a break in the sugar-phosphate backbone.
c. DNA polymerase will not
d. The absence of a 3-OH group on the ddNTP prevents the
addition of another nucleotide.
Concept 11
DNA fingerprinting detects genetic differences among people
by using probes for highly variable regions of chromosomes.
Concept 12
Forward genetics begins with a phenotype and detects and analyzes
the genotype that causes the phenotype. Reverse genetics
begins with a gene sequence and through analysis determines the
phenotype that it encodes.
Concept Check 6
A geneticist interested in immune function induces random
mutations in a number of genes in mice and then determines which of the resulting mutant mice have impaired immune function. This is
an example of
a. forward genetics. c. both forwa
b. reverse genetics.
Concept 13
A transgenic mouse is produced by the injection of cloned DNA
into the pronucleus of a fertilized egg, followed by implantation of
the egg into a female mouse. In knockout mice, the injected DNA
contains a mutation that disables a gene.
Concept 14
Recombinant DNA technology is used to create a wide range of
commercial products, including pharmaceuticals, specialized bacteria,
genetically engineered crops, and transgenic domestic
animals.
Concept 15
Gene therapy is the direct transfer of genes into humans to treat
disease. Gene therapy was first successfully implemented in 1990
and is now being used to treat genetic diseases, cancer, and infectious
diseases.
Concept 16
Both genetic and physical maps provide information about the relative
positions and distances between genes, molecular markers,
and chromosome segments. Genetic maps are based on rates of
recombination and are measured in percent recombination, or
centimo
Concept Check 7
A contig is
a. a set of molecular markers used in genetic mapping.
b. a set of overlapping fragments that form a continuous stretch
of DNA.
c. a set of fragments generated by a restriction enzyme.
d. a small DNA fragment used in sequencing
b. a set of overlapping fragments that form a continuous stretch
Concept 17
Sequencing a genome requires breaking it up into small overlapping
fragments whose DNA sequences can be determined in a
sequencing reaction. In map-based sequencing, sequenced fragments
are ordered into the final genome sequence with the use of
genetic an
Concept 18
The Human Genome Project was an effort to sequence the entire
human genome. Begun in 1990, a rough draft of the sequence was
completed by two competing teams, an international consortium
of publicly supported investigators and a private company, both of
w
Concept Check 8
The Human Genome Sequencing Consortium used which approach
in sequencing the human genome?
a. Whole-genome shotgun sequencing
b. Map-based sequencing
c. A combination of whole-genome shotgun sequencing and
map-based sequencing
b. Map-based sequencing
Concept 19
Genomic projects are collecting databases of nucleotides that vary
among individual organisms (single-nucleotide polymorphisms,
SNPs). Bioinformatics is a interdisciplinary field that combines
molecular biology and computer science. It develops databases
Concept 20
The function of an unknown gene can sometimes be determined
by finding genes with similar sequence whose function is known.
Concept 21
Microarrays, consisting of DNA probes attached to a solid support,
can be used to determine which RNA and DNA sequences are present
in a mixture of nucleic acids. They are capable of determining
which RNA molecules are being synthesized and can thus be us
Concept 22
Comparative genomics compares the content and organization of
whole genomic sequences from different organisms. Prokaryotic
genomes are small, usually ranging from 1 million to 3 million base
pairs of DNA, with several thousand genes.
Concept Check 9
What is the relation between genome size and gene number in
prokaryotes?
Species with larger genomes generally have more genes than
do species with smaller genomes, and so gene density is relatively
constant.
Concept 23
Genome size varies greatly among eukaryotic species. For multicellular
eukaryotic organisms, there is no clear relation between
organismal complexity and amount of DNA or gene number. A
substantial part of the genome in eukaryotic organisms consists of
re
Concept 24
The proteome is the complete set of proteins found in a cell.
Techniques of protein separation and mass spectrometry are used
to identify the proteins present within a cell. Microarrays are used
to determine sets of interacting proteins. Structural proteo