1
Q
recombinant DNA molecules
A
constructed from pieces of naturally occurring chromosomes and plasmids
2
Q
Module 3
polymerase chain reaction (PCR)
A
- repeated copying of a short segment of a DNA molecule
- uses 2 primers
- 3’ end of primers are facing each other
3
Q
The basis of recombinant DNA technology is the ability to manipulate DNA molecules in the test tube. This, in turn, depends on the availability of _____ ______ whose activities are known and can be controlled, and which can therefore be used to make specified changes to the DNA molecules
A
purified enzymes
4
Q
The enzymes available to the molecular biologist fall into four broad categories:
A
- DNA polymerases: synthesize new polynucleotides complementary to an existing DNA or RNA template
- Nucleases: degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next
- Ligases: join DNA molecules together by synthesizing phosphodiester bonds between nucleotides at the ends of two different molecules or at the two ends of a single molecule
- End-modification enzymes: make changes to the ends of DNA molecules
5
Q
Module 3
The new polynucleotide is always synthesized in the _____ direction: DNA polymerases that make DNA in the other direction are unknown in nature.
A
5ʹ → 3ʹ
6
Q
A
7
Q
A
8
Q
Module 3
- DNA polymerase requires a _____ in order to initiate the synthesis of a new polynucleotide.
- The sequence of this primer determines the … and …
- When a DNA polymerase is used to make new DNA in vitro, the primer is usually a ______ _______ made by chemical synthesis.
- The primer is usually ______ nucleotides in length
- 3’ mismatch =
- Mismatches at other positions are _____ and useful for …
- ____ _____ within primer are tolerated, useful for introducing ______ into genes In the same way, deletions in primer are useful for introducing _____ into genes
A
- primer
- position at which it attaches to the template DNA
- specifies the region of the template that will be copied
- short oligonucleotide
- 20
- no PCR product, because DNA polymerase can’t extend
- tolerated
- introducing mutations into genes
- Large insertions
- insertions
- deletions
9
Q
Module 3
the DnA synthesis and exonuclease activities of DnA polymerases
A
- 5ʹ → 3ʹ DNA synthesis activity: enables polymerase to add nucleotides to the 3’-end of the strand that it is synthesizing
- 3ʹ → 5ʹ exonuclease activity: enables polymerase to remove one or more nucleotides from the 3’-end of the strand that it is making (proofreading)
- 5ʹ → 3ʹ exonuclease activity: enables polymerase to remove one or more nucleotides from the 5ʹ-end of a polynucleotide that is already attached to the template strand.
- can degrade the 5ʹ-end of a polynucleotide that has just been synthesized
- enzyme is almost always prepared from E. coli cells whose polymerase gene has been engineered so that the resulting enzyme has the desired properties
- aka Gap Repair
- Usually during synthesis of lagging strand
- Cleavage of the phosphodiester bond of DNA
10
Q
exonucleases
A
removes nucleotides from the ends of DNA and/or RNA molecules
11
Q
endonucleases
A
makes cuts at internal phosphodiester bonds
12
Q
Module 3
restriction endonucleases types
A
- three main types: I, II, III
- With types I and III, there is no strict control over the position of the cut
- with type II the cut is always at the same place, either within the recognition sequence or very close to it
13
Q
restriction endonucleases
A
- enzyme that binds to a DNA molecule at a specific sequence
- makes a doublestranded cut at or near that sequence
- Because of sequence specificity, the positions of cuts within a DNA molecule can be predicted if the DNA sequence is known
14
Q
There are also examples of restriction enzymes with degenerate recognition sequences, meaning that
A
- they cut DNA at any of a family of related sites
- i.e. HinfI recognizes 5ʹGANTC3ʹ, where N is any nucleotide, and so it cuts at 5ʹGAATC3ʹ, 5ʹGATTC3ʹ, 5ʹGAGTC3ʹ, and 5ʹGACTC3ʹ
- Most cut within the recognition sequence, but a few, such as BsrBI, cut at a specified position outside of this sequence
15
Q
Module 2
Restriction enzymes cut DNA in two different ways:
A
- blunt or flush end: simple doublestranded cut
- sticky or cohesive ends:
- cuts the two DNA strands at different positions, usually two or four nucleotides apart
- not good for creating genomic libraries because larger overhangs of at least 15 to 16 bp are needed to ensure a single occurrence in human genome
- resulting DNA fragments have short, singlestranded overhangs at each end
- base pairing between them can stick the DNA molecule back together again
16
Q
One feature that is particularly important in recombinant DNA technology is that some pairs of restriction enzymes have different _____ ______ but give the same _____ _____
A
- recognition sequences
- sticky ends
17
Q
Module 3
gel electrophoresis
A
the standard method for separating DNA molecules of different lengths
18
Q
in gel electrophoresis. _____ _____ is the critical determinant of migration rate. This is because …
A
- molecular length
- the gel is a network of pores through which the DNA molecules have to travel to reach the positive electrode
- Shorter molecules are less impeded by the pores than are longer molecules and so move through the gel more quickly
19
Q
In order to follow the progress of the electrophoresis, one or two _____ of known migration rates are added to the DNA samples before loading. The bands of DNA can be visualized by soaking the gel in _____ ______ solution. This compound ______ between DNA base pairs and ______ when activated with _____ ______
A
- dyes
- ethidium bromide
- intercalates
- fluoresces
- ultraviolet radiation
20
Q
DNA fragments that have been generated by treatment with a restriction endonuclease can be joined back together again or attached to a new partner by a DNA ligase. The reaction requires energy, which is provided by
A
adding either ATP or nicotinamide adenine dinucleotide (NAD) to the reaction mixture, depending on the type of ligase used
21
Q
Module 3
quantitative PCR
A
- enables the amount of target DNA present at the start of the PCR to be measured
- rate of product synthesis during the test PCR is compared with the progress of control PCRs with known amounts of starting DNA
- comparison imade by identifying the stage in the PCR at which the amount of fluorescent signal reaches a preset threshold
- The more rapidly the threshold is reached, the greater the amount of target DNA in the starting mixture
22
Q
Module 3
In a PCR experiment, the target DNA is mixed with
A
- Taq DNA polymerase
- pair of oligonucleotide primers
- nucleotides
23
Q
Module 3
Primers attach to the target DNA at ____ _____ of the segment to be copied. This means that the sequences of these attachment sites must be known so that primers of the appropriate sequences can be _____
A
- either side
- synthesized
24
Q
Module 3
Reaction is started by heating the mixture to ______ so that the
_____ ______ that hold together the two polynucleotides of the double helix are broken, _____ the target DNA into single-stranded molecules
A
- 94°C
- hydrogen bonds
- denaturing
25
# Module 3
The temperature is reduced to \_\_\_\_\_\_, which results in some rejoining of the single strands of the target DNA but also allows the primers to _____ which means to:
* 50–60°C
* anneal
* attach to their positions
26
# Module 3
After primers anneal, DNA _____ begin, so the temperature is raised to \_\_\_\_\_, the optimum temp for _____ \_\_\_\_\_\_. In this first stage of the PCR, a set of long products is synthesized from each strand of the target DNA.
* synthesis
* 72°C
* Taq polymerase
27
# Module 3
As cycles of denaturation–annealing–synthesis are repeated, the long products act as templates for new DNA synthesis, giving rise, in the third cycle, to ____ \_\_\_\_\_, which _____ in an exponential fashion. the short products have _____ as part of the ends of the new PCR product.
* short products
* accumulate
* primer
28
PCR reaction is halted and a sample is analyzed by agarose gel electrophoresis, which will reveal a _____ \_\_\_\_\_\_ if the PCR has worked as expected
* single band
29
real-time PCR
* technique of collecting data throughout the PCR process as it occurs
* combines amplification and detection into a single step
* achieved using a variety of different fluorescent chemistries that correlate PCR product concentration to fluorescence intensity
* characterized by the point in time (or PCR cycle) where the target amplification is first detected
* value of detection is usually referred to as cycle threshold (Ct)
30
cycle threshold (Ct)
* time at which fluorescence intensity is greater than background fluorescence
* the greater the quantity of target DNA in the starting material, the faster a significant increase in fluorescent signal will appear, yielding a lower Ct
31
# Module 3
real-time PCR: flourescent dye approach
disadvantage
* it measures the total amount of double-stranded DNA in the PCR at any particular time
* ds DNA may cause overestimate of the actual amount of the product because primers sometimes anneal to themselves in various nonspecific ways, increasing the amount of double-stranded DNA that is present
32
# Module 3
real-time PCR: flourescent dye approach
* a dye that gives a fluorescent signal when it binds to double-stranded DNA is included in the PCR mixture
* gradual increase in the fluorescent signal given out by the mixture indicates rate
33
# Module 3
real-time PCR: reporter probe
* short oligonucleotide
* gives a fluorescent signal when it hybridizes to the PCR product
* probe hybridizes only to the PCR product
* avoids problems caused by primer–primer annealing
34
# Module 3
Förster resonance energy transfer (FRET)
* makes use of a pair of labels
* fluorescent dye
* compound that quenches the fluorescent signal when brought into close proximity with the dye
* dye is attached to one end of the reporter probe
* quenching compound is attached to other end
* usually no fluorescence because probe is designed so the two ends base-pair to one another, placing quencher next to the dye and quenching the fluorescent signal
* Hybridization between probe and PCR product disrupts base pairing, moving quencher away from the dye and enabling the fluorescent signal to be generated
35
PCR length restrictions
* up to 5 kb can be amplified without too much difficulty
* over 5 kb and up to 40 kb—are possible by modifications of the standard technique
* \> 40 kb are unattainable
36
clone library
* inserted DNA fragments come from different parts of the starting DNA
* if enough clones are obtained, then it is possible to have every part of a genome represented in the library
* starting point for a genome sequencing project
* by sequencing the individual fragments in different clones, the genome sequence can gradually be built
37
# Module 2
transformation
* process by which naked DNA is taken up by a bacterial cell
* not a particularly efficient process
* rate of DNA uptake enhanced by suspending cells in calcium chloride before addition of the DNA and then briefly incubating the mixture at 42°C
* **Small plasmids (\<10 kb) are efficiently transformed, but large plasmids aren’t**
* **large plasmids can be transformed using cosmids**
38
# Module 2
Insertional inactivation
* technique used in recombinant DNA technology
* bacteria carrying recombinant plasmids or a fragment of foreign DNA is made to insert into a restriction site inside a gene to resist antibiotics
* causes gene to turn non-functional / inactivated state
39
# Module 2
\_\_\_\_\_ genomes can also be used as cloning vectors because they too possess origins of replication that enable them to be propagated inside bacteria
Bacteriophage
40
# Module 2
the inability of plasmids to handle DNA fragments greater than about _____ led to the use of _____ as closing vectors. Larger inserts are likely to undergo _____ or interfere with the plasmid _____ \_\_\_\_\_ in such a way that the recombinant DNA molecules become lost from the host cells
* 10 kb
* bacteriophages
* rearrangements
* replication system
41
# Module 2
Replacement vectors
* optional DNA is contained within a stuffer fragment, flanked by a pair of restriction sites
* replaced when the DNA to be cloned is ligated into the vector
42
# Module 2
\_\_\_\_\_ vector has the ability to insert moderate lengths of foreign DNA while _____ vector has the ability to accommodate larger lengths of foreign DNA
* insertion
* replacement
43
# Module 2
insertion vectors
* phage vector that has a restriction site introduced within the phage genome at the site of optional DNA
* 05-11 kb length
* No filler fragment
* Important to create cDNA libraries
* A unique cleavage site is present
44
# Module 2
Replacement Vectors
* phage vector developed from the removal of a middle ‘filler fragment’ region of phage DNA
* 08-24 kb length
* Filler fragment gets replaced by a foreign insert
* Important in creating genome libraries
* Cleavage site contains genes that are not essential for the lytic cycle
45
# Module 2
The first attempts to develop vectors able to handle larger fragments of DNA centered on the bacteriophage called
lambda (λ)
46
# Module 2
The λ genome is linear, but the two natural ends of the molecule have 12-nucleotide single-stranded overhangs, called _____ \_\_\_\_\_, which have complementary sequences and so can _____ to one another.
* cos sites
* base-pair
47
# Module 2
A λ cloning vector can obtained as a _____ molecule that can be manipulated in the test tube in the same way as a _____ and reintroduced into E. coli by \_\_\_\_\_
* circular
* plasmid
* transfection
48
transfection
the term used for uptake of naked phage DNA
49
# Module 2
**in vitro packaging**
Treatment with restriction endonuclease produces the left and right arms, both of which have one _____ end and one end with the 12-nucleotide overhang of the _____ site
* blunt
* cos
50
# Module 2
**in vitro packaging**
The DNA to be cloned is _____ and so is inserted _____ the two arms during the ligation step. The arms also ligate to one another via their cos sites, forming a \_\_\_\_\_
* blunt-ended
* between
* concatemer
51
# Module 2
concatemer
* a long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series
* usally copies of an entire genome linked end to end and separated by cos sites
52
# Module 2
Some parts of the concatemer are in the order left arm-insert DNA-right arm, and if this combination is \_\_\_\_\_-\_\_\_\_\_ kb in length, it will be enclosed inside the ______ by the in vitro packaging mix. In this example, parts of the concatemer made up of left arm ligated directly to right arm, without new DNA, are ____ \_\_\_\_\_ to be packaged. DNA is then extracted from \_\_\_\_\_\_.
* 37–52
* capsid
* too short
* plaques
53
# Module 2
The λ phage particle is ____ kb in length. It can accommodate from ____ to ____ of DNA in its capsid. _____ kb is essential DNA. This means that _____ kb to _____ kb of DNA can be packed into its capsid. Therefore inserts can range from ____ kb to _____ kb.
* 48
* 78%
* 105%
* 30
* 37 (78% × 48)
* 50 (105% × 48)
* 7 (37 - 30)
* 20 (50 -30)
54
# Module 2
cosmids
* plasmids containing one or more cohesive sites (cos sites) of bacteriophage λ DNA
* hybrids between plasmid and phage λ vectors
* designed to clone large fragments of DNA and to grow their DNA as a virus or as a plasmid
* used in homologous recombination between two different plasmids in the same cell and grown in both bacteria and animal cells
* DNA is obtained from colonies rather than plaques
55
# Module 2
once inside the cell, the cosmid cannot direct _____ of new phage particles and instead replicates as a \_\_\_\_\_
* synthesis
* plasmid
56
# Module 2
Bacterial artificial chromosomes, or BACs
BIG BAD BAC
* originally created from the F plasmid of E. coli.
* F plasmid is relatively large & have higher capacity for inserted DNA
* can clone fragments up to 350 kb
* designed so that recombinants can be identified by Lac selection
* most popular vectors for cloning large pieces of DNA
57
# Module 2
Bacteriophage P1
* similar to λ vectors
* based on a deleted version of a natural phage genome
* capacity determined by the size of the deletion and the space within the phage particle: up to 110 kb
58
# Module 2
P1-derived artificial chromosomes, or PACs
combine features of P1 vectors and BACs and have a capacity of up to 300 kb
59
# Module 2
Fosmids
* contain the F plasmid origin of replication and a λ cos site
* similar to cosmids in how they are used and capacity for inserted DNA
* have a lower copy number in E. coli, which means that they are less prone to instability problems
60
# Module 2
shuttle vector
* a vector (usually a plasmid) constructed so that it can propagate in two different host species
* DNA inserted into a shuttle vector can be tested or manipulated in two different cell types
* In order to be maintained in bacterial cells they contain a bacterial origin of replication and antibiotic-resistance gene
61
# Module 2
Genomic Libraries
* Ordered collections of DNA fragments maintained as clones
* must be representative: cannot have missing pieces of the genome
* Libraries of larger fragments are better than smaller fragments
* The cloned DNA should be easily purified for sequencing
62
# Module 2
How many bp in the human genome
3,200,000,000 bp
| (billions)
63
# Module 2
type of vector use differs in _____ size. Fewer clones are needed with ____ \_\_\_\_\_ size. This helps with overcoming the _____ \_\_\_\_ problem.
* insert
* larger insert
* repeating DNA
64
# Module 2
* what formula gives you the amount of genome produced by a vector
* for any library it's necessary to have _____ times the length of the genome to ensure it is \_\_\_\_\_
* genome size ÷ (insert size × # of clones) = genome size needed for a representative genome library
* many
* representative
65
# Module 2
temperate phage
* ability of some bacteriophages (notably coliphage λ) to display a lysogenic life cycle
* Many (but not all) can integrate their genomes into their host bacterium's chromosome, together becoming a **lysogen** as the phage genome becomes a **prophage**
* able to undergo a productive, typically lytic life cycle, where the prophage is expressed, replicates the phage genome, and produces phage progeny, which then leave the bacterium
66
# Module 2
plaque assay
* standard method used to determine virus concentration in terms of infectious dose
* determine the number of plaque forming units (pfu) in a virus sample
* conducted in petri dishes or multi-well plates
* host cells are infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar to prevent the virus infection from spreading indiscriminately
* A viral plaque is formed when a virus infects a cell
* infected cell will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated
* infected cell area will create a plaque (an area of infection surrounded by uninfected cells) which can be seen with an optical microscope or visually
* Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL)
* The pfu/mL result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle
67
# Module 2
The one-step multiplication curve for a bacteriophage population follows three steps:
1. inoculation: virions attach to host cells
2. eclipse: entry of the viral genome occurs
3. burst: sufficient numbers of new virions are produced and emerge from the host cell
68
# Module 2
prophage
the genetic material of a bacteriophage, incorporated into the genome of a bacterium and able to produce phages if specifically activated
69
# Module 2
lysogen
* a lysogenic bacterium or bacterial strain
* bacterial cell which can produce and transfer the ability to produce a phage
* A prophage is either integrated into the host's bacteria's chromosome or more rarely exists as a stable plasmid
* prophage expresses gene(s) that repress the phagee's lytic action, until this repression is disrupted
70
# Module 2
A _____ plaque is a sign of _____ growing
* cloudy
* lysogen
71
# Module 2
Copy number
* the average or expected number of copies per host cell
* Plasmids are either low, medium or high copy number
* Plasmids vary widely in copy number depending on three main factors:
* The ori and its constituents
* The size of the plasmid and its associated insert (bigger inserts and plasmids may be replicated at a lower number as they represent a great metabolic burden for the cell)
* Culture conditions (i.e. factors that influence the metabolic burden on the host).
72
# Module 2
High Copy Number Plasmids are a Problem
* Some DNA fragments, such as repetitive DNA are unstable in bacteria
* The problem arises due to high copy number
73
# Module 3
Primer Extension
DNA synthesis from a single primer
74
# Module 3
Klenow Polymerase
* produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin.
* retains 5' → 3' polymerase activity & 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading
* loses its 5' → 3' exonuclease activity
* cuz 5' → 3' exonuclease activity of DNA polymerase I is unsuitable for many applications, the Klenow fragment can be very useful in research
* can be reintroduced by cleaving polymerase w/previase cylosin (cleaves protein & removes amino terminal domain)
* separate domain from proofreading domain
75
# Module 3
5’-3’ Exo is Problematic for DNA Synthesis Applications, why?
* Because the 5’-3’ exonuclease results in primer degradation
* degrades primer as quickly as it synthesizes
* primer is one of the substrates needed for DNA synthesis
* DNA labeling- results in less labeled product formed
* Polymerase Chain Reaction- less PCR product formed
76
# Module 3
Sequanase polymerase
* T7 DNA polymerase with 100-fold higher processivity
* stays longer on the DNA w/out falling off because it lacks proofreading capabilities
* Processivity the ability for longer continuous DNA synthesis runs
* Why? Lacks 3’ to 5’ exonuclease, synthesizes DNA without pause For DNA sequencing, longer runs are possible
77
# Module 3
Taq polymerase
* Thermally stable so perfect for high temperature sequencing applications and PCR
* Lacks 5’→3’ exonuclease
* Revolutionized PCR, first version of PCR used Klenow, had to be added fresh after each heating cycle
78
# Module 3
Reverse transcriptase
* retro-viral DNA polymerase that uses RNA as template strand to synthesizes DNA
* Used to produce cDNAs from RNA
* RNA → DNA → cDNA
Molecular Genetics
flashcards
Decks in class (12)
# Cards
-
Chapter 2: Studying DNA78
-
Chapter 365
-
Chapter 4: Sequencing Genomes39
-
Chapter 5: Genome Annotation74
-
Chapter 6: Identifying Gene Function72
-
Chapter 12: Transcriptomes6
-
Chapter 7: Eukaryotic Nuclear Genomes44
-
Chapter 8: Genomes of Prokaryotes and Eukaryotic Organelles23
-
Chapter 10: Accessing the Genome45
-
Chapter 11: Assembly of the Transcription Initiation Complex77
-
Chapter 12: Synthesis and Processing of Bacterial RNAs35
-
Chapter 13: Synthesis and Processing of the Proteome16
