Chapter Ten: Digital Analysis of DNA Flashcards
___ fragment the genome at specific sites
restriction enzymes
each restriction enzyme recognizes a ___
specific sequence of bases
restriction enzymes cut ___
sugar-phosphate backbones of both strands
___ are generated by digestion of DNA with restriction enzymes
restriction fragments
___ is a commonly used restriction enzyme
EcoR1 from E. coli
restriction enzyme cuts are straight through both strands of DNA at the line of symmetry
blunt ends
restriction enzyme cuts are displaced on either side of the line of symmetry
sticky ends
sticky ends have either 5’ or 3’ ___
overhangs
sticky ends are good for when you want to make ___
recombinant DNA
average restriction fragment size is ___
256 base pairs
___ can be used to fragment DNA at random locations
mechanical forces
mechanical forces can break ___
phosphodiester bonds
two forms of mechanical forces
passing DNA through a thin needle at high pressure
sonication (ultrasound energy)
___ distinguishes DNA fragments according to size
gel electrophoresis
DNA has a ___ charge
negative
___ fragments move further through gel
small
a means to purify a specific DNA fragment away from all other fragments and make many copies of the fragment
molecular cloning
two basic steps of molecular cloning
- insert DNA fragments into coning vectors to specialized chromosome-like carriers that ensure transport, replication, and purification of DNA inserts
- transport recombinant DNA into living cells to be copied
a group of replicated DNA molecules
DNA clone
three main features of a plasmid cloning vector
- origin of replication
- selectable marker gene
- synthetic polylinker
DNA sequence containing multiple restriction enzyme sites
synthetic polylinker
___ and ___ are alternate cloning vectors that can carry large inserts
bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC)
create ___ using plasmid vectors
recombinant DNA
digestion of the vector and human genomic DNA with a ___ results in ___
restriction enzyme
complementary sticky ends
___ is used to deal the phosphodiester backbones between vector and inserted fragment
DNA ligase
process by which a cell or organism takes up foreign DNA
transformation
only cells that ___ will grow on media specific to the selectable marker gene included in the plasma
took up the plasmid
long-lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector
genomic library
a perfect genomic library has one copy of ___
every sequence in the entire genome
___ to obtain a perfect library
impossible
Sanger sequencing uses ___ to make new DNA
DNA polymerase
DNA polymerase requires these 4 things
- template strand to copy
- deoxyribonucleotide triphosphates
- primer
- buffer
a ___ is a good template for Sanger sequencing
recombinant plasmid
cloned recombinant DNA is ___
denatured
strands are mixed with ___
oligonucleotide primer
as temperature is lowered, primers and template strand ___
anneal (hybridize)
hybridized template and primer are mixed with ___, ___, and ___
DNA polymerase, dNTPs, and small amounts of dideoxyribonucleotide triphosphates (ddNTPs)
ddNTPs each have a unique ___
fluorescent tag
ddNTPs lack a ___, so once they are added to the new DNA strand ___
3’ OH
polymerization stops
result of this is a set of ___ with different 3’ ends
nested fragments
nested fragments are separated by size using ___
electrophoresis
___ is recorded to identify nucleotide code
color of terminal ddNTP
E. coli have ___ chromosome, ___ genes, and a genome size of ___
1, 4400, 4.6 Mb
yeast have ___ chromosomes, ___ genes, and a genome size of ___
16, 6000, 12.5 Mb
humans have ___ chromosomes, ___ genes, and a genome size of ___
23, 2800, 3300 Mb
the human genome project began using the ___
hierarchical strategy
steps of hierarchical strategy of sequencing genomes
- construct BAC genomic library
- identify sets of overlapping BAC clones
- shear DNA from each BAC separately to make smaller clones
- sequence DNA based on overlap
steps of whole-genome shotgun strategy for genome sequencing
- create genomic library of overlapping fragments in plasmid vectors
- sequence DNA inserts of randomly chosen library plasmids
- assemble sequences based on overlap of sequences into contigs
the whole genome shotgun approach can be highly ___
automated
sequencing a BAC clone library rather than smaller inserts of plasma clones
paired-end sequencing
trying to figure out what genes code for
annotation
