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


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