13'8 What are the steps that are needed to convert mRNA into cDNA

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13.8 What are the steps that are needed to
convert mRNA into cDNA? How would you obtain
radioactive cDNA? How would you obtain
radioactive mRNA? To convert mRNA into cDNA, the
cells must be broken open (lysed), followed by
the purification of the mRNA from total RNA using
the polyA tail added during the processing of
mRNA. The mRNA is then incubated with reverse
transcriptase, some primer (either oligo dT
or random oligos) and deoxynucleotides (dATP,
dCTP, dTTP, and dGTP). Technically a DNA
polymerase is now required to make the
second strand of cDNA, although for PCR one
strand of cDNA is enough. To make the cDNA
radioactive, simply include one or more
nucleotides with 32P in the a position (closest
to the sugar). To make radioactive mRNA, the
cells or tissue used to make the mRNA must be
incubated with 32P labeled nucleotides before
isolation.
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13.14 What methods are used to get foreign DNA
into eukaryotic cells? What is transfection?
What is a transgenic mouse? Foreign DNA can be
introduced into eukaryotic cells by many
methods including in lipid vesicles
(lipofection), electroporation, direct
injection (very difficult), viruses (both DNA and
RNA based), calcium phosphate precipitation
(somewhat outdated). Transfection is the process
of putting foreign DNA into a cell via chemical
means. Transduction brings in foreign DNA via a
virus. A transgenic mouse is a mouse embryo that
has foreign DNA stuck into its genome. They are
typically made by transfecting embryonic
stem cells with a plasmid DNA and then selecting
for clones that incorporate the DNA into their
genome. They may also be made by viral
infection of the stem cells. The most advanced
transgenic mice are made by using a plasmid DNA
that is similar to part of the mouse genome, and
then incorporating the plasmid in that locus by
homologous recombination.
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13.18 How are DNA fingerprints used in forensic
cases? Could they be used in paternity
exclusion? DNA fingerprinting uses several
polymorphic regions of the human genome that
have different lengths based on the allele. Each
person has only 2 of the number of possible
alleles and may be homozygous or heteroxygous.
By comparing a person's DNA to that obtained
elsewhere, a person can be positively excluded
from having committed a crime if the polymorphic
bands are different sizes. It may only (very
strongly) suggest the a person is likely to have
committed a crime. It could also be used for
paternity exclusion, but in this case only 1
band in the father and one band in the child
needs to be the same size. It can only be used
to exclude a person from being the father or
(strongly) suggest the person is the father.
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13.24 A linear DNA molecule cut with EcoR1 gives
fragments of 3, 4.2, and 5 kb. What are the
possible restriction maps. The linear molecule
must contain 2 EcoR1 sites to give 3 fragments.
The 3kb piece could be found either on the left,
center, or right side. The other two pieces
could be found in one of 2 possible locations for
each position, or 3x26 possible maps. From
probability theory, this is the same as 3! for
the number of possible ways to arrange 3
different items.
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13.26 A 12kb DNA cut with EcoR1 yields one 12kb
band. When the original molecule is cut with
BamH1, 3 fragments of 2, 4.5 and 5.5 kb are
produced. When the DNA is cut with both enzymes
4 bands of 2, 2.5 3.0 and 4.5 kb are produced.
Draw a restriction map of this DNA. Because the
double digest gives 4 bands while cutting with
BamH1 gives 3 bands, there must be one EcoR1
site. Because cutting with just EcoR1 gives one
12kb band, the DNA must be circular- ie. cut it
once, it gives a single linear band. When the
double digest is performed, the 2 and 4.5 kb
bands are not changed and the 5.5 kb band is
replaced by 2.5 and 3.0 kb bands. Thus there are
3 BamH1 sites on the circular molecule, and the
EcoR1 site is found 2.5kb from the nearest BamH1
site within the 5.5 kb BamH1 piece.
2.5
BamH1 site
3
EcoR1 site
2.0
12 kB total length
4.5
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13.34 What is the sequence of the dideoxy
sequencing gel to the right?
To read the gel, the smallest pieces must
correspond to the 5' end as all polymerases work
in the 5' to 3' direction. The ddCTP lane will
give a band every time a C is added. Same for
each other band. The overall sequence
is 5'-CAATAGGTCGAGGTTCAATGG-3'
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13.38 A DNA strand with the sequence
3'-GACTATTCCGAAAC-5' is sequenced by the dideoxy
method. If the reaction mixture contains all
4 radioactive deoxynucleotide triphosphates
(dNTP) plus dideoxythymidine what size labeled
bands do you expect to see on the gel? The newly
synthesized DNA strand will be made 5' to 3' (ie.
left to right!) Dideoxythymidine will cause the
polymerase to stop every time a T is added, so it
will stop when it encounters an A on the
complimentary strand. Thus it will stop at
positions 2, 5, 11, 12, and 13 on the given
sequence some percentage of the time, but there
are many newly synthesized strands so that
statistically you will find bands at all
positions.