Genetics 10.1-10.2 Smartbook
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1. Isolate genomic DNA 2. Make a genomic library of 500-1000bp fragments from the entire genome in plasmid vectors 3. Sequence the inserts of randomly chosen library plasmids 4. Use a computer to identify overlaps between DNA sequences and assemble progressively larger contigs
Compared to a deoxyribonucleotide triphosphate (dNTP), a dideoxyribonucleotide triphosphate (ddNTP) is missing its ____.
3' hydroxyl group
Which of the following are requirements of DNA polymerase in order for it to synthesize DNA?
A short oligonucleotide primer, A single-stranded template DNA molecule, Deoxyribonucleotide triphosphates
The enzyme that catalyzes DNA replication is called _____ _____.
DNA polymerase
What technique would a scientist use to separate the mixture of DNA fragments produced during a Sanger sequencing reaction?
Polyacrylamide gel electrophoresis
What process is used in the whole-genome shotgun sequencing strategy?
Random clones in a genomic library are sequenced, and a computer is used to assemble the entire genome based on sequence overlaps.
What process is catalyzed by the enzyme DNA polymerase?
Replication of DNA
Sanger sequencing generates a nested array of fragments. What two features of the fragments are used to determine the sequence of nucleotides in the template DNA?
The length of a fragment, The terminal 3' base
In a DNA sequencing reaction, the template DNA molecule and the primer interact by forming ______.
complementary base pairs
A nucleotide that lacks both the 2' and 3' hydroxyl groups is called a(n) _____.
dideoxynucleotide
In automated sequencing, each dideoxyribonucleotide is labeled with a different colored ______.
fluorescent dye
In Sanger sequencing, a double-stranded template DNA molecule is made single-stranded by subjecting it to ____.
heat
Two single-stranded DNA molecules can form a double helix if they contain complementary sequences. This process is called ______.
hybridization
In a Sanger sequencing reaction, different DNA fragments in a nested array can be distinguished from one another based on their length and the _____.
identity of the fluorescent dideoxynucleotide at the 3' end
Detection of the products of a Sanger sequencing reaction requires the use of a(n) _____ that will stimulate the fluorescence of the dyes attached to the dideoxynucleotides.
laser
Sanger sequencing generates many fragments that differ in length by a single nucleotide. Such a set of fragments is known as a _____ _____.
nested array
Because a dideoxynucleotide does not have a 3' hydroxyl group, when it is added to a growing DNA strand, ______.
no additional nucleotides can be added to the new DNA strand
One type of genome shotgun sequencing uses paired-end sequencing which refers to ______.
obtaining 2 sequence reads from a BAC clone, one from each end of the insert
When Celera scientists sequenced the human genome, they used randomly selected BAC clones and used each random BAC clone to obtain 2 sequences, one from each end of the insert. This approach is known as _____-_____ sequencing.
paired, end
The products of a Sanger sequencing reaction can be separated from one another through the use of _____ gel electrophoresis.
polyacrylamide
In the context of a genome sequencing project, the term shotgun means that the fragments that are sequenced are chosen from a library _____.
randomly
In a DNA sequencing reaction, the DNA is heated to a high temperature in order to _____.
separate the two strands of the template DNA from one another
The use of dideoxyribonucleotides with different colored fluorescent dyes allows the detection of the ______.
sequence of DNA
A genome sequencing approach in which the clones that are sequenced are selected from a library at random is called a(n) _____ strategy.
shotgun
In order to catalyze DNA replication, DNA polymerase requires a single-stranded DNA molecule that can act as a(n) ___ deoxyribonucleotide ___ to serve as the building blocks for newly synthesized DNA, and a short oligonucleotide ____ with a free 3' end.
template, triphosphate, primer
When a dideoxynucleotide is incorporated into a growing DNA strand, the new DNA strand is terminated because _____.
the absence of the 3' hydroxyl group prevents DNA polymerase from attaching another nucleotide to the new DNA strand
Sanger sequencing produces a nested array of DNA fragments. These DNA fragments have ______.
the same 5' end, but different 3' ends
To analyze the DNA fragments produced by a Sanger sequencing reaction, the products would be separated by polyacrylamide gel electrophoresis and then detected by ______.
using a laser to stimulate fluorescence of the labeled dideoxynucleotides
