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Blueprint Series

Complete Solution
Chapter 6

6
MOLECULAR
GENETIC TECHNIQUES

REVIEW THE CONCEPTS
1
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Recessive alleles usually result from a mutation that
inactivates the affected gene, leading to loss of function
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Lethal recessive mutations can be maintained in heterozygotes
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Dominant alleles often result from a mutation that causes
some kind of gain of function
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Thus, researchers may use conditional
mutations such as temperature sensitive mutations to study the effects of dominant
lethal alleles
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A temperature sensitive mutation is one where the gene is non-functional at a given
temperature, usually a lower or higher than normal growth temperature
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3
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If a heterozygous organism containing
both mutations shows the mutant phenotype, then the two mutations are in the same
gene because neither allele provides a functional copy of the gene
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Dominant
mutations cannot be tested by complementation analysis because they will display a mutant phenotype even in the presence of a wild-type allele of the gene
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She will grow them on media without lysine
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She will know her mutant has been complemented when
she sees a colony that is now white (the wild-type color)
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Bacteria that synthesize restriction enzymes also synthesize a DNA modifying
enzyme to protect its own DNA
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Methylated DNA is no longer a substrate for the
encoded restriction enzyme
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After being cut with a restriction enzyme, the
ends of the cut DNA molecule can exist as single-stranded tails (sticky ends) with
either 5’ or 3’ overhangs, or as blunt (flush) ends
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6
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A plasmid is useful for cloning DNA
fragments up to approximately 20 kb
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7
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A cDNA
(complementary DNA) library is a collection of DNA molecules that are copied
from messenger RNA molecules using the enzyme reverse transcriptase
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In contrast, a genomic DNA library consists
of random fragments of the total genome
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You could use either of the
genomic libraries or the cDNA library from neurons
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If the
skin cell does not express Gene X, that cDNA will not be present in the library
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The PCR reaction is performed as multiple cycles of a three-step process
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The second step
involves cooling the DNA solution to allow annealing of short single-stranded
oligonucleotide primers that are complementary to the target DNA molecule
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The resultant double-stranded DNA molecules are then subjected to
further rounds of denaturation, annealing, and DNA synthesis (extension)
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A nonthermostable DNA polymerase would be inactivated by heat denaturation
during each cycle of the PCR process and would necessitate the addition of new
enzyme prior to each DNA synthesis step
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Southern blotting is a technique in which DNA fragments are separated by size
in a gel and then transferred to a solid support such as a nitrocellulose or nylon
membrane
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The hybridized probe is then detected by some
technique such as autoradiography
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Southern blotting can be used to identify a DNA fragment that contains a
DNA sequence of interest
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10
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A promoter that is inducible would provide even higher expression levels of the foreign gene product
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An example of this type of molecular tag is a short
sequence of histidine residues (a polyhistidine sequence)
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Other
proteins can be washed out and the His-tagged protein can be released from
the nickel atoms by lowering the pH of the solution
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These posttranslational modifications are essential for
the biological activity of the recombinant protein
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Northern blotting and RT-PCR are useful for analyzing fewer genes because
they rely on the creation of specific radioactive probes or PCR primers
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Studying
entire genomes (or chromosomes in this example) is most easily accomplished
with a microarray, which uses a chip that can hybridize to hundreds or thousands of genes based on complementary base pairing
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The expression of mRNA in individual cells can be determined by in situ hybridization in whole cells or tissue sections
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After washing to
remove excess probe, the cells can be examined microscopically to detect the
locations of labeled mRNA
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13
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Simple-sequence repeats (SSRs), also known as microsatellites, consist of a variable number of repeating one-, two-, or three-base
sequences
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Both types of polymorphisms can be used as molecular
markers for mapping studies
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In general, the farther two markers are separated on a chromosome, the greater the recombination frequency between those two markers,
and vice versa
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Linkage disequilibrium mapping can sometimes be used in cases where
a genetic disease commonly found in a particular population results
from a single mutation that occurred many generations in the past
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The closer
genetic markers are to each other, the less likely they will be recombined
by crossing over during meiosis
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DNA
polymorphisms on part of a chromosome that are inherited together are
called haplotypes
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15
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One
strategy for iden-tifying a disease gene involves gene expression
analysis
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Northern blot analysis allows a
comparison of both the level of expression and the size of the transcripts
between normal and disease tissues
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In this case, DNA sequencing of a potential disease gene from
tissues of anormal and a disease state could reveal a single nucleotide
change that results in the disease phenotype
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To generate a knockout mouse, mouse embryonic stem cells are first
transfectedwith a disrupted allele of the target gene
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The ES cells, which now contain a
mutant gene, are injected into a blastocyst
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Pups that are born
will be chimeras
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Using the above technology, loxP sites can be
engineered to flank the gene of interest
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Knockout mice serve as models for human diseases
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17
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This type
of mutation pro- duces a loss of function phenotype
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In this method, a short double-stranded RNA
molecule is introduced into cells
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