Chapter 10 (book) Flashcards
1
Q
The entire complement of information contained within the DNA sequences of an organism is known as the
A
- Genome
2
Q
Genomics
A
the study of whole genomes.
3
Q
Enzymes that bind to a specific sequence in a double-stranded DNA molecule and cut the DNA backbone of each strand are called
A
- Restriction enzymes
- bacterial proteins that recognize specific, short nucleotide sequences and cleave the DNA backbone at those sites.
4
Q
restriction fragments
A
- DNA fragments generated by the action of restriction enzymes
5
Q
In genetics, the term []refers to cutting a DNA molecule with a restriction enzyme
A
- Digestion: the enzymatic process by which a complex biological molecule (DNA, RNA, protein, or complex carbohydrate) is broken down into smaller components.
6
Q
What name is given to the entire complement of genetic material in an organism?
A
- Genome
7
Q
The function of a restriction enzyme that occurs naturally in a bacterial cell is to
A
- protect the cell from viruses
- The restriction enzymes digest viral DNA to protect prokaryotic cells from viral infection
- Bacteria shield their own genomes from digestion by these restriction enzymes through the selective addition of methyl groups to the restriction recognition sites in their genomic DNA. In the test tube, restriction enzymes from bacteria recognize target sequences of four to eight base pairs (bp) in DNA isolated from any other organisms and cut the DNA at or near these sites.
8
Q
Protruding single-stranded overhangs of DNA created by restriction enzymes are called
A
- sticky ends
- sticky ends: the result achieved after digestion by a restriction enzyme that breaks the phosphodiester bonds on the two strands of a DNA molecule at offset locations. The resulting double-stranded DNA molecule has a single protruding strand at each end that is usually one to four bases in length.
9
Q
Restriction enzymes usually recognize [] sequences in which the sequence in one strand is identical to the complementary strand read in the opposite direction.
A
- Palindromic
- Palindromic symmetry is where the base sequences of each of the two DNA strands are identical when read in the 5 to 3 direction. Base pairs, usually 4-6, on either sides of a central line of symmetry are mirror images of each other. Each enzyme always cuts at the same place relative to its specific recognition sequence.
- Palindromic sequence: A DNA sequence in one strand that is identical when read in the opposite direction in the other strand is called.
10
Q
A DNA fragment in which the end of the molecule does not have an overhang on either the 5’ or 3’ strand is said to have []
A
- Blunt ends
- blunt end: an end of a double-stranded DNA molecule that has no 5’ or 3’ overhang.
- If a restriction enzyme cuts a DNA molecule at the line of symmetry within its recognition sequence, it will produce DNA fragments with blunt ends
11
Q
Restriction enzymes typically recognize palindromic DNA sequences. Which of the following sequences is an example of such a sequence?
A
- The palindromic sequence should be read identical to the top strand if read backwards
- Ex: 5’-ACTAGT-3’ 3’-TGATCA-5’ are palindromic because if you read the second strand backwards, it is the same as the first.
- If you only have one strand, you need to compose the complementary strand to see if it is palindromic. If the complementary strand is the same as the original strand backwards, the sequence is palindromic.
12
Q
Many species of bacterial cells make restriction enzymes to protect themselves from invasion by
A
- Viruses
13
Q
What assumptions are made in order to estimate the average length of the fragments that will be produced by digestion of a piece of DNA with a restriction enzyme?
A
- The four bases occur in equal proportions.
- The bases are randomly distributed in the DNA sequence.
14
Q
Formula for calculating average size of the restriction fragments that will be produced by digestion of genomic DNA.
A
- 4^number of bases
15
Q
To calculate the number of restriction fragments produced by a treating the genome with a specific enzyme:
A
- The number of base pairs in the genome/the average number of base pairs the enzyme cuts
- Ex: Enzyme cuts: 65500 bp, Genome: 3billion bp; Number of restriction fragments produced: About 46,000 (3billlion/ 65500)
16
Q
The fraction of viral particles that enter and replicate inside host bacterial cells is called the [] efficiency.
A
- Plating
- Plating efficiency: the fraction of viral cells entering and replicating inside the host bacterial cells
17
Q
What is meant by the term restriction?
A
- The ability of a bacterial cell to prevent the replication of a virus
- Restriction: the bacterial capacity for limiting viral growth.
18
Q
The phenomenon in which growth on a restricting host results in modification of a virus so that succeeding generations grow more efficiently on the same host strain is called?
A
- Modification
- Modification: the phenomenon in which growth on a restricting host changes a phage so that succeeding generations grow more efficiently on that same host.
19
Q
A modification enzyme is a bacterial enzyme that:
A
- adds methyl groups to specific DNA sequences
- modification enzymes: enzymes that add methyl groups to certain bases within specific DNA sequences, preventing the action of particular restriction enzymes on that DNA.
- In a restriction modification system, the term modification refers to modification of a virus, resulting in improved growth on the same host in subsequent generations
20
Q
Shearing of DNA by mechanical stress, such as sonication or passing DNA through a needle at high pressure, cuts DNA []
A
- Randomly
- Random cutting of DNA can be achieved by subjecting the molecules to mechanical stress such as passing the sample through very thin needles at high pressure or by sonication (the application of ultrasound energy). By pulling different parts of the DNA molecule in different directions, these mechanical forces can break phosphodiester bonds at random positions thus fragmenting the DNA in the sample.
21
Q
A lab technician using ultrasound to shear DNA accidentally used a level of ultrasound energy higher than she intended. What result would this mistake have on the size of the DNA fragments produced by ultrasound treatment?
A
- The DNA fragments would be smaller than expected.
- When using mechanical stress researchers can obtain fragments of specific average sizes by changing the amount of mechanical stress. Higher energy ultrasound produced smaller fragments.
22
Q
In order to increase the size of DNA fragments produced by sonication, one would [] the energy of the ultrasound used.
A
- decrease
23
Q
The technique that is used to separate charged molecules based on their movement in an electric field is called []
A
- Electrophoresis
24
Q
A researcher who wanted to break DNA at random locations would most likely subject the DNA to []
A
- mechanical shearing forces
25
DNA fragments that are subjected to electrophoresis will always move toward the positively charged electrode because the [] groups in the backbone of DNA are negatively charged.
- Phosphate
- Because all the phosphate groups in the backbone of DNA carry a net negative charge in a solution near neutral PH, DNA molecules are pulled through the gel toward the wire with a positive charge.
26
What variables affect the rate at which molecules move during electrophoresis?
- The physical size of the molecule
- The composition of the gel
- The strength of the electric field that is applied
27
During electrophoresis, the rate at which different linear DNA molecules will move through a gel is determined by their _____.
- Sizes
- The only one of the variables differing among linear DNA is a particular gel is size. The reason is all the same charge density (because the charge of all nucleotide pairs is nearly identical). As a result, only differences in size cause different linear DNA molecules to migrate at different speeds during electrophoresis.
28
Gel Electrophoresis
- Separates DNA fragments according to their sizes. The smaller the fragment the faster it will migrate in the gel towards the positive electrode.
29
To determine the distance DNA fragments will travel from the well the [] number of base pairs the greater it will travel
- Shorter
30
Electrophoresis involves the movement of charged molecules in a [] field
- Electric
31
After electrophoresis, DNA may be incubated with a compound called ethidium bromide in order to
- make the DNA fragments in the gel visible under ultraviolet light
- If the 4th step in gel electrophoresis
32
During electrophoresis, DNA fragments move toward the electrode with a [] charge because the phosphate groups in the backbone of DNA each carry a(n) []
- Positive; negative
33
After electrophoresis, the size of a DNA fragment can be estimated by comparing the distance the DNA fragment migrated to the distance traveled by a set of [] fragments
- Marker
34
A researcher would most likely choose to use a polyacrylamide gel rather than an agarose gel in order to _____.
- distinguish DNA fragments with small size differences
35
When a collection of DNA fragments is separated by electrophoresis, different DNA fragments will migrate at different speeds due to differences in their []
- sizes
36
A carrier DNA molecule that allows a DNA fragment of interest to be transported into a host cell, replicated, and then purified is called a
- cloning vector
- definition: vehicles for introducing foreign DNA into host cells, where that DNA can be reproduced in large quantities; a DNA molecule into which another DNA fragment of appropriate size can be integrated without loss of the vector’s capacity for replication.
- Can be used to introduce a DNA fragment of interest into a host cell that can replicate it.
37
molecular cloning (definition + purpose)
- the process by which a single DNA fragment is purified from a complex mixture of DNA molecules and then amplified into a large number of identical copies.
- Purpose: To produce many copies of a DNA molecule of interest
- The process of isolating a single fragment of DNA from a complex mixture and making many exact copies of it in a living cell
38
What is the purpose of including marker fragments in a lane of an electrophoresis gel?
- The distance traveled by the marker fragments can be used to estimate the sizes of other fragments
39
To distinguish DNA fragments whose size differences are hundreds or thousands of base pairs, a researcher would most likely electrophorese them using a gel made of
- Agarose
40
Two steps of molecular cloning
- (1) insert DNA fragments into cloning vectors; (2) introduce cloning vectors with DNA inserts into a living cell
41
DNA clone
- a purified sample containing a large number of identical DNA molecules.
- A group of identical DNA molecules
- A group of replicated DNA molecules that are exact copies of one another
42
DNA library
- A collection of cloned DNA molecules possibly purified for immediate study or stored within cells or viruses as collections of clones for future analysis.
43
recombinant DNA molecules
- a combination of DNA molecules with parts having different origins that were joined using recombinant DNA technologies.
- a vector and an inserted fragment
44
2 specialized DNA sequences a Vector must have to be used as a cloning vector
- A sequence that allows bacteria transformed with the vector to be detected by the investigator
- A sequence that allows the vector, and the foreign DNA inserted in it, to replicate
45
Advantage of sticky DNA ends compared to blunt ends in molecular cloning
- Single-strand overhangs are available for base pairing.
- No matter what the origin of the DNA is, two sticky ends produced with the same enzyme are always compatible meaning they are complementary in sequence.
46
DNA ligase
- covalently links the sugar-phosphate backbone of DNA.
- Stabilizes the molecule by forming phosphodiester bonds between adjacent nucleotides (one from the vector and one rom the genomic DNA insert)
47
genomic library
A collection of vectors where restriction enzymes digest the DNA and DNA ligases the vectors to create DNA clones. There is a representative copy of every DNA sequence of the genome of a particular organisms.
48
Plasmid
- A small circular double stranded DNA molecule that can replicate within bacterial cells and is often used as a vector in gene cloning.
49
Recombinant DNA molecule
- A molecule that has covalently linked DNA fragments from at least two sources
50
Requirements for a plasmid vector useful for cloning + why they are required
- origin of replication: short sequence of nucleotides where DNA replication initiates. Required so the DNA can replicate independently/ be copied many times by replication enzymes of the cell .
- a site that can be recognized and cut by a restriction enzyme
- a gene that confers resistance to an antibiotic. This allows the vector to survive in a medium containing a specific antibiotic. The researcher can select the bacterial cells only containing the plasmid.
51
A vector has only two specialized DNA sequences: one that gives it the ability to replicate in E. coli cells and one that allows scientists to insert foreign DNA into it. Can this vector be used as a cloning vector by a scientists working with human DNA?
- No, because it will be impossible to detect cells that have acquired the vector
52
Plasmid vectors can usually carry only relatively short fragments up to
- 20 kb long
53
selectable markers (definition + why they are used)
- vector genes that make it possible to identify cells harboring a recombinant DNA molecule.
- Used to identify cells containing the vector
54
Artificial chromosome (definition + purpose)
- recombinant DNA molecules combining replication and segregation elements. They behave like normal chromosomes when introduced into a host cell.
- Use to clone a large piece of DNA
55
Two specialized DNA sequences a vector must have in order to be used as a cloning vector
- A sequence allowing the vector, and inserted foreign DNA to replicate
- A sequence allowing bacteria transformed with the vector to be detected by the investigator
56
Treatment of bacterial cells with cold CaCl2 or subjecting them to electric shock can be used to increase the likelihood that [] will occur [blank answer and why]
- Transformation because it increases the permeability of the bacterial cell membrane, creating temporary holes the DNA can enter through.
57
To clone a DNA molecule, a researcher would digest the DNA of interest and a vector with the same [] enzyme, then join the two molecules together with the enzyme DNA [] finally, the recombinant molecule is introduced into bacteria using a process called [].
- Restriction; ligase; transformation
58
Role of the polylinker in a plasmid that will be used as cloning vector
- It contains a number of different restriction sites
- Polylinker: a synthetic DNA sequence in a cloning vector containing several different restriction enzyme recognition sites that can be used for insertion of a DNA molecule.
59
Transformation
- process in which cells take up foreign DNA molecules
60
If bacteria are transformed with a plasmid carrying an antibiotic-resistance gene, one would expect progeny of that cell to ______ when exposed to the antibiotic.
- Grow
61
Vectors that can be used to clone large segments of DNA
- BAC, YAC
62
Cellular clone (definition + what it is made of)
- a group of cells that all descend from a single progenitor cell.
- A cellular clone is made up of genetically identical bacterial cells
DNA clone is made up of identical molecules of a(n) plasmid within the colony.
63
Steps involved in cloning a recombinant DNA molecule:
The DNA of interest and a vector are digested with the same restriction enzyme
DNA ligase is used to joining the DNA of interest and a vector
The recombinant DNA molecule is transformed into bacterial cells
Bacterial cells are grown on medium containing a selectable marker, such as a antibiotic.
64
Ways that will increase the permeability of the cell membrane to DNA
- suspension in a cold CaCl2 solution
| - applying a high-voltage electric shock
65
A complete genomic library
A single copy of every sequence of an organism's genome
| 4-5 genomic equivalents is preferable in a library.
66
Genomic equivalent
- clones present in a library containing exactly one copy of every DNA sequence from an organism's genome.
- the number of different DNA clones—with inserts of a particular size— required to carry a single copy of every sequence in a particular genome.
67
Two types of clones transformed with foreign DNA and allowed to grow into a colony
- cellular clone: The colony as a whole
| - DNA clone: The many plasmid molecules within the colony
68
Formula to determine genomic equivalent
- Nucleotides/ kb of the genome/ average insert size
69
DNA polymerase
- The enzyme that catalyzes DNA replication
70
Requirements of DNA polymerase in order for it to synthesize DNA
- A short oligonucleotide primer; has a free 3’ end
- Template: A single-stranded DNA molecule
- Deoxyribonucleotide triphosphates: serve as the building blocks for newly synthesized DNA (dCTP, dGTP, dTTP)
71
In a DNA sequencing reaction, the template DNA molecule and the primer interact by forming
- complementary base pairs
72
In Sanger sequencing, a double-stranded template DNA molecule is made single-stranded by subjecting it to ____.
- Heat
| - The heat disrupts the hydrogen bonds to melt the DNA into a single strand
73
Two features of the fragments are used to determine the sequence of nucleotides in the template DNA in sanger sequencing.
- The terminal 3' base
| - The length of a fragment
74
Dideoxynucleotide + different colored fluorescent dyes + why new strand is terminated
- A nucleotide that lacks both the 2' and 3' hydroxyl groups
- allows the detection of the sequence of DNA
- the absence of the 3' hydroxyl group prevents DNA polymerase from attaching another nucleotide to the new DNA strand
75
Hybridization (hybridize)
- base pairing between a probe and its complementary DNA sequence.
- base pairing between a probe and its complementary DNA sequence.
76
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
77
Nested array
- Set of fragments that differ in length by a single nucleotide generate by Sanger sequencing.
78
Polyacrylamide gel electrophoresis + how DNA fragments are detected
- Can separate the products of a Sanger sequencing reaction
| - use a laser to stimulate fluorescence of the labeled dideoxynucleotides
79
In automated sequencing, each dideoxyribonucleotide is labeled with a different colored ______.
- fluorescent dye
80
Nested array of DNA fragments produced by Sanger sequencing
- the same 5' end, but different 3' ends
81
Whole-genome shotgun sequencing
- a strategy for determining an entire genome sequence where random overlapping genomic DNA fragments are sequenced, and the sequences are assembled by a computer until the entire genome sequence is complete.
- Random clones in a genomic library are sequenced, and a computer is used to assemble the entire genome based on sequence overlaps.
82
In the context of a genome sequencing project, the term shotgun means that the fragments that are sequenced are chosen from a library _____.
- Randomly
83
Paired end sequencing
- a strategy for determining the base pair sequence of whole genomes in which ∼1000 bp at both ends of single BAC clones are sequenced. Knowing that the two sequence reads are connected on a single BAC insert allows genome assembly despite the presence of repeated elements.
- obtaining 2 sequence reads from a BAC clone, one from each end of the insert
- Done by Celera
84
Steps involved in whole-genome shotgun sequencing
- isolate genomic DNA
- Make genomic library of 500-1000bp fragments from the entire genome in plasmid vectors
- Sequence the inserts of randomly chosen library plasmids
- Use a computer to identify overlaps between DNA sequences and assemble progressively larger contigs/ contugous fragments or fragments with larger overlaps.
