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DNA manipulation techniques Flashcards

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1
Q

What are DNA manipulation techniques used for?

A
  • Use of enzymes to manipulate DNA
  • Synthesis of DNA using polymerase
  • Joining DNA with ligase
  • Cutting DNA with endonucleases

These techniques are fundamental for genetic engineering and biotechnology applications.

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2
Q

What is the function of CRISPR-Cas9 in bacteria?

A

Editing an organism’s genome

CRISPR-Cas9 is a revolutionary tool for precise genetic modifications.

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3
Q

What is the purpose of polymerase chain reaction (PCR)?

A

Amplification of DNA

PCR is a technique used to make multiple copies of a specific DNA segment.

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4
Q

What role does gel electrophoresis play in DNA analysis?

A

Sorting DNA fragments

It is used for interpreting gel runs for DNA profiling.

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5
Q

What are recombinant plasmids used for?

A

Transforming bacterial cells

They are vectors that can carry foreign DNA into host cells, such as in the production of human insulin.

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6
Q

What is the purpose of using genetically modified organisms (GMOs) in agriculture?

A
  • Increase crop productivity
  • Provide resistance to disease

GMOs are engineered to enhance agricultural efficiency and sustainability.

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7
Q

Define genetically modified organisms (GMOs).

A

Organisms whose genomes have been altered using genetic engineering technology

This includes a wide range of organisms modified for various purposes.

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8
Q

What distinguishes transgenic organisms from GMOs?

A

Alteration involves genetic material from a different species

Transgenic organisms are a specific subgroup of GMOs.

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9
Q

Give examples of genetically modified organisms.

A
  • Salt-tolerant wheat
  • Bt. cotton
  • Golden rice

These examples illustrate the application of genetic modification in agriculture.

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10
Q

What is the function of restriction enzymes?

A

To cut DNA at specific sequences

Restriction enzymes are essential tools in molecular biology for DNA manipulation.

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11
Q

What is the function of DNA ligase?

A

To join DNA fragments together

DNA ligase is crucial for DNA replication and repair processes.

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12
Q

What are restriction enzymes also known as?

A

restriction endonucleases

They occur naturally in bacteria and cleave foreign DNA from invading viruses.

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13
Q

What is the primary function of restriction enzymes?

A

To cut DNA in a precise way

They enable specific cleavage of DNA at recognition sequences.

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14
Q

What is a recognition sequence in the context of restriction enzymes?

A

Usually between 4 and 8 bases long

Each restriction enzyme has a different recognition sequence.

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15
Q

What type of ends do blunt-end restriction enzymes leave?

A

Clean cut ends

They cut the sugar-phosphate backbone on both strands of DNA at the same location.

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16
Q

What type of ends do sticky-end restriction enzymes leave?

A

Overhanging ends

They cut the sugar-phosphate backbone at different locations on each strand.

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17
Q

Fill in the blank: Blunt-end restriction enzymes leave _______ ends.

A

clean cut

This results from cutting the sugar-phosphate backbone at the same location on both strands.

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18
Q

Fill in the blank: Sticky-end restriction enzymes leave DNA fragments with _______ ends.

A

overhanging

This occurs because they cut at different locations on each strand.

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19
Q

What is DNA ligase?

A

An enzyme that facilitates bonding between two fragments of DNA

DNA ligase plays a crucial role in DNA replication and repair.

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20
Q

What does CRISPR stand for?

A

Clustered Regularly Interspaced Short Palindromic Repeats

A short section of naturally occurring viral DNA that is inserted into a bacterial chromosome and used to prevent future infections.

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21
Q

What is the role of the Cas9 enzyme in the CRISPR system?

A

Cuts DNA at the target site

The Cas9 enzyme is guided by RNA to make precise cuts in the DNA for gene editing.

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22
Q

What is the function of guide RNA (gRNA) in CRISPR gene editing?

A

Directs Cas9 to a specific DNA sequence

The gRNA is complementary to the target DNA of interest.

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23
Q

What happens when Cas9 encounters the bacterium’s own DNA?

A

It does not cut the DNA

The Cas9 enzyme is specific to certain sequences of viral DNA.

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24
Q

What is the process called when the cell repairs DNA breaks using a provided template?

A

Homology-Directed Repair (HDR)

This process is used to insert a new gene or sequence during gene editing.

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25
What is the process called that introduces small insertions or deletions (indels) during DNA repair?
Non-Homologous End Joining (NHEJ) ## Footnote This is an error-prone process that can disrupt the gene's sequence.
26
What is a **PAM** in the context of CRISPR?
A short sequence recognized by Cas9 ## Footnote PAM is typically the sequence 'NGG' that allows Cas9 to cut upstream of it.
27
What is the purpose of the **CRISPR array** in bacteria?
Acts as a memory bank for viral infections ## Footnote Snippets of viral DNA are pasted into palindromic sequences within the bacterial genome.
28
What is the outcome of a **gene knock out** using CRISPR?
Disruption of the gene's sequence ## Footnote This is achieved through small insertions or deletions introduced during DNA repair.
29
What is the outcome of a **gene knock in** using CRISPR?
Insertion of a new gene or sequence ## Footnote This is facilitated by providing a DNA template during the HDR process.
30
What does **tracrRNA** do in the CRISPR system?
Binds to the repeats and assists in RNA processing ## Footnote It is complementary to the repeats in the CRISPR array.
31
True or false: The CRISPR-Cas9 system functions naturally in bacteria to provide immunity against viral infections.
TRUE ## Footnote The system enables bacteria to destroy viral DNA sequences and prevent subsequent infections.
32
What is formed when a virus infects a bacterial cell in the context of CRISPR?
A CRISPR locus ## Footnote This locus contains snippets of viral DNA that serve as a memory for future infections.
33
What is the **single-guide RNA (sgRNA)** in CRISPR gene editing?
A synthesized RNA that is complementary to the target DNA ## Footnote It is a fusion of the crRNA and tracrRNA regions.
34
What technique is used to **amplify DNA**?
Polymerase Chain Reaction (PCR) ## Footnote PCR allows researchers to create vast quantities of DNA identical to trace samples.
35
List some **procedures in DNA technology** that require substantial amounts of DNA.
* DNA sequencing * DNA profiling/fingerprinting * Gene cloning * Making artificial genes ## Footnote These procedures are essential for various applications in genetics and forensic science.
36
What is the purpose of a **thermal cycler** in PCR?
To amplify DNA ## Footnote Thermal cyclers are simple-to-use machines that facilitate the PCR process.
37
What components are placed in the **thermal cycler** for PCR?
* DNA sample * Primers * Free nucleotides * Buffer solution * DNA polymerase ## Footnote These components are essential for the amplification process.
38
What happens to DNA during the **denaturation** step of PCR?
DNA strands are separated by heating the sample to 95°C for 5 minutes ## Footnote This step is crucial for allowing the primers to bind to the target DNA.
39
What is the role of **primers** in PCR?
To anneal (bond) to the DNA ## Footnote Primers are short strands of mRNA that initiate the synthesis of new DNA strands.
40
What is the **cooling temperature** for the sample during PCR?
60°C ## Footnote Cooling allows the thermally stable DNA polymerase enzyme to bind to the primers.
41
What does the **extension** step in PCR involve?
Synthesis of a complementary strand of DNA using free nucleotides ## Footnote This step is facilitated by the DNA polymerase enzyme.
42
Remember the acronym **D.A.E** for the steps in PCR: D stands for _______.
Denature ## Footnote The acronym helps to recall the sequence of steps in the PCR process.
43
Remember the acronym **D.A.E** for the steps in PCR: A stands for _______.
Anneal ## Footnote This step involves the bonding of primers to the separated DNA strands.
44
Remember the acronym **D.A.E** for the steps in PCR: E stands for _______.
Extension ## Footnote This step involves synthesizing new DNA strands complementary to the target DNA.
45
After one cycle of PCR, how many copies of the original DNA sample are there?
Two copies ## Footnote Each cycle of PCR doubles the amount of DNA present.
46
What is **gel electrophoresis** used for?
To separate large molecules (including nucleic acids or proteins) based on their size, electric charge, and other physical properties ## Footnote Gel electrophoresis is a common technique in molecular biology.
47
What is a **restriction digest**?
A process that cuts DNA into smaller pieces, producing a range of DNA of different lengths ## Footnote This step is crucial for preparing DNA for electrophoresis.
48
What is the role of the **buffer solution** in gel electrophoresis?
To cover the DNA samples placed in wells ## Footnote The buffer solution maintains the pH and provides ions for conducting electricity.
49
In gel electrophoresis, where are the DNA samples placed?
In **wells** created in the gel ## Footnote Wells are holes made with a comb before the gel solidifies.
50
What charge do DNA fragments carry?
Negatively charged ## Footnote The negative charge is due to the phosphates in the DNA backbone.
51
What happens to DNA fragments in gel electrophoresis when an **electric field** is applied?
They move towards the positive terminal ## Footnote Smaller fragments move faster than larger ones.
52
What does the **gel matrix** do in gel electrophoresis?
Acts as a sieve for the DNA molecules ## Footnote It helps separate molecules of different sizes.
53
What are **DNA markers** used for?
To estimate the sizes of the DNA fragments in the sample lanes ## Footnote They consist of a mixture of DNA molecules with known molecular weights.
54
True or false: Larger DNA fragments move faster than smaller ones in gel electrophoresis.
FALSE ## Footnote Smaller fragments move faster than larger fragments due to the gel matrix.
55
What is the purpose of applying **dyes or radio-labelled markers** in gel electrophoresis?
To visualize the separated DNA fragments ## Footnote This allows for the analysis of the DNA after electrophoresis.
56
What is the application of **DNA profiling**?
Identifying individuals ## Footnote DNA profiling is used in forensic science, paternity testing, and genetic research.
57
How are **short tandem repeats** used in DNA profiling?
To construct DNA profiles ## Footnote Short tandem repeats are regions in DNA where a short sequence of bases is repeated, and variations in these repeats can help distinguish individuals.
58
What does **DNA Profiling** compare for the identification of individuals?
Variable short tandem repeat (STR) regions of the genome ## Footnote DNA profiling is a technique used in forensic science and paternity testing.
59
At each gene locus, one **STR** is inherited from _______.
either parent ## Footnote This inheritance pattern is crucial for individual identification.
60
What do **STRs** consist of?
* A variable number of tandem repeats * A 2 to 6 base pair sequence ## Footnote STRs are used in DNA profiling due to their variability among individuals.
61
In the example shown, a two-base sequence **CA** is repeated. This is an example of a _______.
short tandem repeat (STR) ## Footnote STRs are important markers in genetic analysis.
62
What is **recombinant DNA**?
DNA produced when DNA from two sources are recombined ## Footnote This process allows for the joining of DNA fragments from different sources.
63
What must be done to **plasmid DNA** and **foreign DNA** to create a **recombinant plasmid**?
Both must be cut with the same **restriction enzyme** ## Footnote This creates complementary **sticky-ends** for joining.
64
What is the process called when the two matching **sticky-ends** of DNA come together?
Annealing ## Footnote This involves joining by complementary base pairing.
65
What are **plasmids**?
Small circular pieces of DNA found in **prokaryotes** ## Footnote Plasmids can be used to transfer genetic material.
66
What happens after the two different DNA fragments are cut using the same **restriction enzyme**?
They are attracted to each other by weak **hydrogen bonds** ## Footnote This attraction facilitates the joining of DNA fragments.
67
What is the **second step** in the creation of a recombinant plasmid?
Joining the complementary bases of the two pieces of DNA ## Footnote This step follows the initial attraction of the DNA fragments.
68
Fill in the blank: The joined fragments will form either a **_______** molecule or a circular one.
linear ## Footnote This refers to the possible shapes of the recombinant DNA.
69
What is the role of **restriction sites** in the process of creating recombinant DNA?
They are attracted by **base pairing** only ## Footnote This specificity is crucial for the correct joining of DNA fragments.
70
What must be done to **plasmid DNA** and **foreign DNA** to create a **recombinant plasmid**?
Both must be cut with the same **restriction enzyme** ## Footnote This creates complementary **sticky-ends** for joining.
71
What is the process called when the two matching **sticky-ends** of DNA come together?
Annealing ## Footnote This involves joining by complementary base pairing.
72
What are **plasmids**?
Small circular pieces of DNA found in **prokaryotes** ## Footnote Plasmids can be used to transfer genetic material.
73
What happens after the two different DNA fragments are cut using the same **restriction enzyme**?
They are attracted to each other by weak **hydrogen bonds** ## Footnote This attraction facilitates the joining of DNA fragments.
74
What is the **second step** in the creation of a recombinant plasmid?
Joining the complementary bases of the two pieces of DNA ## Footnote This step follows the initial attraction of the DNA fragments.
75
Fill in the blank: The joined fragments will form either a **_______** molecule or a circular one.
linear ## Footnote This refers to the possible shapes of the recombinant DNA.
76
What is the role of **restriction sites** in the process of creating recombinant DNA?
They are attracted by **base pairing** only ## Footnote This specificity is crucial for the correct joining of DNA fragments.
77
What enzyme is responsible for joining DNA fragments together in the creation of recombinant DNA?
DNA ligase ## Footnote DNA ligase links fragments permanently, producing a molecule of recombinant DNA.
78
What is the **third step** in the creation of a recombinant plasmid?
Ligation ## Footnote This step involves joining the sugar phosphate backbone of the two pieces of DNA.
79
What process is used to insert a recombinant plasmid into a bacterial cell?
Transformation ## Footnote This process allows the bacteria to express a new gene and acquire new characteristics.
80
What happens to bacteria when a foreign plasmid is incorporated into them?
They are said to be ‘transformed’ ## Footnote Transformed bacteria can express new genes and exhibit new traits.
81
Name the two methods of **artificial bacterial transformation** used in biotechnology.
* Heat shock * Electroporation ## Footnote These methods facilitate the uptake of plasmids by disrupting the plasma membrane of bacterial cells.
82
What is commonly used to select transformed bacterial cells during the transformation process?
Antibiotic selection ## Footnote The plasmid vector contains an antibiotic resistance gene, allowing only transgenic cells to grow in the presence of antibiotics.
83
What is **Bt cotton** engineered for?
Resistant to insects ## Footnote This genetic modification increases crop yield and reduces insecticide use by farmers.
84
What characteristic does **salt tolerant wheat** possess?
Survives high salinity soils ## Footnote This engineering leads to increased crop yield.
85
What is the purpose of **Golden rice**?
Contains higher levels of vitamin A ## Footnote It can help prevent vitamin A deficiency in humans.
86
What is the benefit of **Roundup ready canola**?
Resistant to glyphosate herbicides ## Footnote Farmers can spray for weeds without harming the canola plants.
87
True or false: Not every bacterial cell takes up the recombinant plasmid during the transformation process.
TRUE ## Footnote This necessitates a selection method to identify successful transformations.
88
Describe the function of the PAM sequence adjacent to the sgRNA target sequence?
Pam sequece is required for CAS-9 enzyme recognition and binding to the DNA, where as CAS-9 will only cut the DNA if the PAM sequence is present to make sure correct targeting and prevernting cutting off CRISPR sequecne .
89
Outline the function of the CRISPR DNA sequence in prokaryotes?
The CRISPR DNA sequence provides adaptive immunity in prokaryotes by protecting them against bacteriophage viruses, so the spacer sequences are derived from foreign viral DNA. These spacers are transcribed into RNA, which guides Cas protein to recognise and cut matching viral DNA during future infections
90
role of DNA ligase ?
DNA ligase joins the dna fragments together by developing a phosphodiester bond in the sugar phosphate after the DNA is editied.
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