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Quiz 1 Flashcards

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1
Q

Human Genome Project

A
  • an accurate sequence of the
    human genome was initiated in 1990 an completed
  • (97%
    coverage) in 2003
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2
Q

General ideas behind genome sequencing are simple

A
  • Fragmenting the genome
  • Cloning DNA fragments
  • Sequencing DNA fragments
  • Reconstructing the genome sequence from fragments
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3
Q

What do restriction enzymes do?

A

They fragment the genome at specific sites by cutting the sugar-phosphate backbones of both DNA strands.

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4
Q

What is the average restriction fragment size for a 6-base recognition site?

A

Approximately 4100 bp (4.1 kb)

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5
Q

What are the recognition sites for restriction enzymes?

A

4 to 8 bp of double-stranded DNA

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6
Q

Palindromic

A

base sequences of each strand are identical when read 5′-to-3’

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7
Q

What are the two types of ends produced by restriction enzymes?

A

Blunt and sticky ends

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8
Q

Blunt ends

A

cuts are straight through both DNA strands at the line of symmetry

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9
Q

Sticky ends

A

cuts are displaced equally on either side of line of symmetry

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10
Q

What is gel electrophoresis used for?

A

To distinguish DNA fragments according to size.

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11
Q

What is the general formula for fragment length?

A

4n, where n is the number of bases in the recognition site

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12
Q

Where does 4-base recognition occur?

A

Every 4^4 bp
Average restriction fragment size is 256 bp

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13
Q

Where does 6-base recognition occur?

A

Every 4^6 bp
Average restriction size is 4100 bp (4.1 kb)

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14
Q

What is molecular cloning?

A

A method to purify a specific DNA fragment away from all other fragments and make many identical copies.

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15
Q

Where can mechanical forces be used?

A

They can be used to fragment DNA at randome locations

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16
Q

How do mechanical forces break phosphodiester bonds?

A
  • passing DNA through a thin needle at high pressure
  • sonication (ultrasound energy)
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17
Q

Electrophoresis

A

Movement of charged molecules

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18
Q

Step 1 to Gel Electrophoresis

A

Pour heated molten agarose into an acrylic plate to which a comb has been attached, allow to cool and harden

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19
Q

Step 2 to Gel Electrophoresis

A

Remove comb, place gel in buffered aqueous solution, load DNA samples into wells in gel

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20
Q

Step 3 to Gel Electrophoresis

A

Apply electric current
- DNA has negative charge, so move towards postive charge

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21
Q

Step 4 of Gel Electrophoresis

A

Remove gel from tank after electrophoresis

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22
Q

Step 5 of Gel Electrophoresis

A

Visualize DNA fragments by staining gel with fluorescent
dye, photograph gel under U V light

  • With linear DNA fragments, migration distance through gel
    depends on size
  • Determine size of unknown fragments by comparison of migration to DNA markers of known size
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23
Q

What is molecular cloning used for?

A

Analyzation of genomes of animals, plants, and microorganisms

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24
Q

Steps of Molecular Cloning

A

1) insertion of DNA fragments into cloning vectors to specialized
chromosome-like carriers that ensure transport,
replication, and purification of DNA inserts
2) transport recombinant DNA into living cells to be copied

Group of replicated DNA molecules = DNA clone

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25
What are the three main features of plasmid cloning vectors?
Origin of replication, a selectable marker gene, and a synthetic polylinker.
26
What is the purpose of the origin of replication in plasmid vectors?
It allows for the replication of the plasmid within the host cell.
27
What is a selectable marker gene?
A gene that provides a way to identify cells that have successfully taken up the plasmid, often by conferring antibiotic resistance.
28
What is a synthetic polylinker?
A DNA sequence containing multiple restriction enzyme sites that facilitates the insertion of DNA fragments.
29
What are bacterial artificial chromosomes (BAC)?
Cloning vectors that can carry large inserts of DNA, approximately 300 kb.
30
What are yeast artificial chromosomes (YAC)?
Cloning vectors that can carry very large inserts of DNA, approximately 2000 kb.
31
How large is a typical plasmid insert?
Approximately 20 kb.
32
What does the digestion of the vector and human genomic DNA do?
Included with a restriction enzyme, this would result in a complementary sticky ends
33
What is used to seal the phosphodiester backbones between vectors?
DNA ligase is used to seal both vector and inserted fragements
34
What is transformation in molecular cloning?
The process by which a cell or organism takes up foreign DNA.
35
What percentage of E. coli cells typically get transformed with plasmid DNA?
Only 0.1% of E. coli cells.
36
On what type of media will only the cells with plasmid grow?
Media containing ampicillin.
37
What happens to each E. coli cell that successfully takes up a plasmid?
Each cell produces a colony on an agar plate.
38
What does a colony of transformed E. coli cells represent in terms of plasmids?
The millions of identical plasmids in that colony form a DNA clone.
39
Genomic libarary
- long-lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector - each colony contains a different recombinant plasmid, each with a part of the human genome
40
What defines a perfect genomic library?
A perfect genomic library has one copy of every sequence in the entire genome.
41
Genomic equivalent
The number of clones in a perfect library that represents the entire genome.
42
Why is it impossible to obtain a perfect genomic library?
Because it is challenging to ensure one copy of every sequence in the entire genome.
43
How many genomic equivalents are typically made in a genomic library?
Usually four to five genomic equivalents.
44
What is the probability that each locus is present at least once in a typical genomic library?
95% probability.
45
What are the roles of DNA polymerase, template, and primer in a Sanger sequencing reaction?
DNA polymerase synthesizes new DNA using the template strand and primer.
46
What is the equation for # of clones in perfect library?
length of genome over average size of inserts
47
What does DNA polymerase require?
template, Deoxyribonucleotide triphosphates, and primer
48
Template
single strand of DNA to copy
49
Deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP),
basic building blocks for new DNA
50
Primer
short single stranded DNA molecule that is complementary to part of the template, includes free 3’ end to which DNA polymerase can attach new nucleotides
51
Why is a recombinant plasmid considered a good template for Sanger sequencing?
It contains cloned recombinant DNA that can be sequenced.
52
What process is used to prepare cloned recombinant DNA for Sanger sequencing?
Denaturation, where heat breaks the hydrogen bonds between strands.
53
What is mixed with the denatured strands during Sanger sequencing?
An oligonucleotide primer approximately 20 bp long that is complementary to the template strand.
54
What happens as the temperature is lowered in the Sanger sequencing process?
The primers and template strand anneal (hybridize).
55
What does Sanger sequencing generate?
A series of single-stranded DNA fragments.
56
What components are mixed with the hybridized template and primers in Sanger sequencing?
DNA polymerase, dNTPs, and small amounts of dideoxyribonucleotide triphosphates (ddNTPs).
57
What is the significance of ddNTPs in Sanger sequencing?
They lack a 3′ –OH group, which halts polymerization.
58
What is the result of incorporating ddNTPs during the sequencing process?
A set of nested fragments, each with a different 3′ end.
59
What terminates DNA synthesis?
incorporating dideoxynucleotide (ddNTP)
60
What are nested fragements separated by?
Size using electrophoresis
61
What seperates DNA fragments that differ in size by one nucleotide?
A special gel does this - smaller DNA fragments migrate quicker and appear at the bottom of the gel
62
How is each ddNTP labeled for detection in Sanger sequencing?
Each ddNTP is labeled with a different fluorescent dye.
63
What does each lane in a sequencing gel represent?
The sequence obtained from a separate DNA sample and primer.
64
How does each fragment in Sanger sequencing terminate?
Each fragment terminates with a specific ddNTP labeled with a unique fluorescent dye.
65
What method is used to separate DNA fragments in Sanger sequencing?
Electrophoresis based on size.
66
How is the nucleotide code identified in Sanger sequencing?
By recording the color of the terminal ddNTP.
67
What happens to fragments in automated DNA sequencing?
Fragments flow past a laser beam and the color of the terminal base is digitally recorded
68
What does a DNA sequence trace represent in automated Sanger sequencing?
It shows the inferred DNA sequence complementary to the template strand.
69
How is the sequence read in Sanger sequencing?
From left to right.
70
In which direction is the DNA synthesized during Sanger sequencing?
5′ to 3′ synthesis from the primer.
71
What role does the computer play in interpreting Sanger sequencing results?
The computer reads the sequence complementary to the template strand.
72
What happened during the Human Genome Project for hierachcial strategy?
construct BAC genomic libarary, identity sets of overlapping BAC clones, shear DNA from each BAC separately to make similar clones, sequence DNA, assemble sequences based on overlap
73
What strategy did Celera develop for genome sequencing?
The whole-genome shotgun strategy.
74
What is the first step in the whole-genome shotgun strategy?
Create a genomic library of overlapping fragments in plasmid vectors.
75
What does the "shotgun" approach involve in this sequencing strategy? second step
Sequencing DNA inserts of randomly chosen library plasmids.
76
How are sequences assembled in the whole-genome shotgun strategy? third step
Based on the overlap of sequences into contigs—continuous base pair sequences.
77
Each shotgun clone...
was partially sequences from both ends
78
Paired-end sequencing
sequencing a BAC clone library rather than smaller inserts of plasma clones
79
What does each BAC clones insert have three pieces of?
- Two approximately 1000 bp sequence read * Knowledge that the two sequences were approximately 200 to 300 kb apart

Genetics (1 decks)

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