3A4 Laboratory Technology Flashcards
Describe basic laboratory techniques like gel electrophoresis, PCR, and gene therapy.
1
Q
What is a microscope?
A
An optical instrument used for viewing very small objects.
2
Q
What is microscopy?
A
The act of using a microscope.
3
Q
What are the three main types of microscopy?
A
- Optical
- Scanning probe
- Electron
4
Q
When was optical microscopy invented?
A
17th century
5
Q
What are some examples of optical microscopes?
A
- Simple microscopes
- Compound microscopes
Other examples include:
- Stereoscopic microscopes
- Metallographic microscopes
- Polarizing microscopes
- UV light microscopes
6
Q
How do optical microscopes magnify images?
A
By using visible light that passes through or reflects off the specimen and lenses to magnify the image.
7
Q
What is a simple microscope?
A
Uses one lens to view objects at a higher magnification.
8
Q
What is a compound microscope?
A
Uses multiple lenses to increase both the magnification and resolution of the specimen for closer examination.
9
Q
How do scanning probe microscopes work?
A
They move a physical probe across the surface of an object to feel its various shapes and imperfections.
Scanning probe microscopes are used to scan atoms of objects on the nanoscale.
10
Q
What is the probe tip size in scanning probe microscopes?
A
As small as one atom.
11
Q
How does electron microscopy create images?
A
By firing electrons at an object.
Can be used to produce extremely high-resolution images of both biological and nonbiological objects.
12
Q
What is the benefit of using an electron microscope?
A
Electrons have a wavelength 100,000 times smaller than light, increasing the resolution of the image.
13
Q
How do electron microscopes differ from optical microscopes?
A
Electron microscopes bounce electrons off an object instead of light, allowing for visualization of much smaller things.
14
Q
What are the two methods of electron microscopy?
A
- Transmission electron microscopy (TEM)
- Scanning electron microscopy (SEM)
15
Q
What type of microscope is used to understand the chemical make-up of an object?
A
Scanning transmission electron microscope
(STEM)
16
Q
What is a confocal microscope used for?
A
To produce a sharp image for analyzing crystal structures in biological materials.
17
Q
What is the main purpose of an acoustic microscope?
A
To detect faults in manufactured objects.
18
Q
Define gel electrophoresis.
A
A laboratory method using materials like agarose, polyacrylamide, and starch to separate molecules such as DNA, RNA, and proteins based on their lengths.
19
Q
How does gel electrophoresis work?
A
By loading DNA samples into wells and applying an electric current to move the molecules from the negative to positive electrode for size measurement.
20
Q
What are the different types of gel electrophoresis?
A
- Agarose gel electrophoresis: measures DNA fragment lengths.
- Polyacrylamide gel electrophoresis: analyzes RNA fragments and separates single proteins.
- Starch gel electrophoresis: used for studying DNA and protein polymorphisms.
21
Q
What is agarose gel electrophoresis?
A
A laboratory method that involves the usage of agarose to separate fragments of DNA, RNA, or proteins according to their size.
22
Q
How do DNA fragments move through agarose gel?
A
Based on their size, with smaller fragments moving faster than larger ones.
23
Q
What is agarose gel made from?
A
A gel form purified from seaweed that forms small horizontal holes or pores allowing fragments of DNA, RNA, or protein to migrate through.
24
Q
What is the role of restriction enzymes in genetic engineering?
A
Excising specific genes from the genome.
25
What is the function of **DNA ligase** in genetic engineering?
Gluing genes into a plasmid.
26
What is the main function of the **power supply** in gel electrophoresis?
To provide controlled voltage and amperage to the system.
27
What is the purpose of **combs** in agarose gel electrophoresis?
To embed wells into the agarose gel for loading molecules.
28
What does the **dye front** indicate in gel electrophoresis?
The dye line that migrates the fastest, it provides a visual to ensure that the sample does not migrate off the end of the gel.
29
What is used to visualize stained molecules in gel electrophoresis?
UV box
| (ultraviolet transilluminator)
30
What is the **process** of gel electrophoresis?
1. Gel preparation.
2. Loading samples and standards.
3. Running electrophoresis.
4. Visualizing samples in the gel.
5. Documenting results in the gel.
31
What is the function of **electrophoresis buffer** in DNA electrophoresis?
To complete the electrical circuit between the positive and negative electrodes.
32
How are DNA fragments **separated based on size** in agarose gel electrophoresis?
Shorter DNA molecules travel through agarose faster than longer DNA molecules.
33
What is the purpose of **loading buffer** in DNA electrophoresis?
To add color and density to DNA samples for insertion into the wells in the gel.
34
What is **ethidium bromide** used for in DNA electrophoresis?
To **visualize DNA fragments** by fluorescing when exposed to ultraviolet light.
35
What are the **4 main components** of gel electrophoresis?
* Agarose gel
* Gel box and power supply
* Loading buffer and dye front
* Ethidium bromide and UV box
## Footnote
These components work together to complete the electrophoresis process.
36
What is a **DNA ladder** used for in gel electrophoresis?
To **determine the size of DNA fragments** in an electrophoresis gel.
37
What can be concluded by **comparing the bands** in an electrophoresis gel to DNA ladder standards?
The size of the starting sample DNA and the product of a restriction digest.
38
What does the presence of a **3 kb band** in a gel electrophoresis lane indicate?
Incomplete digestion, where uncut DNA is present.
39
What is the purpose of using gel electrophoresis in the analysis of a **ligation reaction**?
To determine if the pieces of DNA have been successfully ligated together.
## Footnote
If they have been successfully ligated, a new 3 kb band will be formed.
40
What is a **spectrophotometer**?
An instrument that **measures the transmission or absorption of light** through a sample substance.
41
What are the **parts** of a spectrophotometer?
* Light source
* Monochromator
* Cuvette
42
What is the wavelength range for **visible light**?
Between 380 to 740 nanometers.
43
What are the **two light sources** in a spectrophotometer?
* Tungsten lamp (visible light)
* Deuterium or hydrogen lamp (ultraviolet range)
44
What is the purpose of setting the **'blank'** in a spectrophotometer?
To ensure that the absorption of the cuvette and liquid themselves is not added to the absorption of the sample alone.
45
What material can the **cuvette** of a spectrophotometer be made of?
* Glass
* Silica
* Plastic
* Quartz
46
Why must the cuvette be free of fingerprints or oils before inserting it into the spectrophotometer?
To ensure accurate readings.
47
What is the function of the **monochromator** in a spectrophotometer?
It splits the light into its components, producing light of a **single wavelength.**
48
What type of spectrophotometer is designed to **break down the sample into ions**?
Atomic absorption spectrophotometer
49
What does a **photocell** do in a spectrophotometer?
Converts the light passing through the sample into electrical energy.
50
What can be calculated from the absorption reading in a spectrophotometer?
The concentration of the compound in the sample.
51
What is **Beer's Law**?
A = Ebc
## Footnote
Where:
A = Absorption (no units)
E = Epsilon, or molar absorptivity, or extinction coefficient with units L mol-1
b = The path length of the sample, or width of the cuvette, in centimeters (cm)
c = concentration of the compound in solution, in mol/L
52
What is **Polymerase Chain Reaction**?
| (PCR)
A nucleic acid amplification technique that **amplifies genetic material several millions to billions of times**.
53
What are the **five basic reagents** used in PCR?
* Template DNA
* PCR primers
* Nucleotides
* PCR buffer
* Taq polymerase
54
What is the function of **template DNA** in PCR?
It is the **starting material for genetic amplification** and is the piece of DNA that PCR copies.
55
What is the role of **PCR primers** in the PCR process?
They are short segments of single-stranded DNA that bind to the template DNA and **identify the regions** targeted for amplification during PCR.
56
What is the role of **Taq polymerase** in PCR?
Creates copies of DNA and is heat resistant.
57
Why is Taq polymerase used in PCR instead of human DNA polymerase?
Because Taq polymerase is heat stable and **can withstand high temperatures**.
## Footnote
Ideal temperature for Taq polymerase to function is between 70 and 80°C.
58
What are the **three primary steps** of PCR?
1. Denaturation
2. Annealing
3. Extension
59
What is the purpose of the **denaturation** step in PCR?
To break the hydrogen bonds between complementary base pairs, and unwind DNA.
## Footnote
Denaturation should occur at 95°C for 30 seconds.
60
What is the purpose of the **annealing** step in PCR?
To allow primers to attach to the DNA template.
## Footnote
Annealing should occur at 50°C for 30 seconds.
61
What is the purpose of the **extension/elongation** step in PCR?
To amplify a certain region of DNA by adding nucleotides.
## Footnote
Extension should occur at 72°C for 1 minute.
62
What is the purpose of the **amplification** step in PCR?
To **repeat the denaturation, annealing, and extension steps** for up to 35 cycles to create multiple copies of DNA.
63
What are **PCR buffers** used for?
To keep **pH stable** and assist Taq polymerase with synthesizing DNA.
64
What are **Deoxynucleotide triphosphates (dNTPs)** in PCR?
Free nucleotides of adenine (A), thymine (T), cytosine (C), and guanine (G) required for Taq polymerase to synthesize new DNA.
65
What is the role of the **thermocycler** in the PCR process?
To cycle between different temperatures over a set period of time for the denaturation, annealing, and extension steps.
66
Why is it important for PCR temperatures to **change at each step**?
To facilitate the denaturation, annealing, and extension steps of the PCR process.
67
What is the function of **DNA primase** in PCR?
**Adds RNA primers** to each strand of DNA.
68
Which enzyme attaches to the primers and adds complementary nucleotides to make a new copied strand of DNA in PCR?
DNA polymerase
69
What is the process of repeating the denaturation, annealing, and extension steps of PCR known as?
PCR cycling
70
What is the purpose of using a **PCR machine** in the PCR process?
To **automatically change the temperature** of the PCR tubes during cycling.
71
What is a **genome**?
A complete code or set of an organism's DNA.
## Footnote
The human genome contains about 30,000 genes.
72
What was the primary objective of the **Human Genome Project**?
To complete DNA sequencing of the entire human genome.
## Footnote
Approximately 3 billion base pairs.
73
What method of DNA sequencing was first developed by **Fred Sanger** in 1977?
The Sanger method.
## Footnote
This was the method of DNA sequencing utilized by the Human Genome Project.
74
How is the **Sanger method** used for DNA sequencing?
By copying the DNA repeatedly and finding the location of each nucleotide base.
75
What are some **applications** of DNA sequencing?
* Determining ancestry
* Disease risk assessment
* Crime scene analysis
76
Why is DNA sequencing **important**?
It allows us to better understand human traits, heredity, and disease.
77
What is **dye terminator sequencing?**
A method of DNA sequencing where each base is tagged with a special color of fluorescent dye, and a sequencing machine reads the DNA sequence by the order of colors.
## Footnote
Much faster and cheaper than the Sanger method of DNA sequencing.
78
What is **gene therapy** and how is it accomplished?
A therapeutic technique that **replaces damaged proteins** by inserting the DNA for that protein into a cell.
## Footnote
Proteins are responsible for cellular functions, and diseases can result from improper protein production.
79
What are the **cons** of gene therapy?
* Not possible when the genetic code for a protein is unknown.
* The DNA introduced will degrade naturally inside the cell, leading to transient treatment.
* The patient's immune system may recognize the DNA as foreign and attack it, potentially harming the patient.
* Prohibitively expensive.
80
What are the **pros** of gene therapy?
* Enables production of dysfunctional proteins inside a cell.
* Can be tailored to replace any protein, making it useful for various diseases.
81
What is the **history** of gene therapy?
* The concept of gene therapy was introduced in 1972.
* The first human trial occurring in 1990.
82
What is an example of **common** gene therapy treatment?
Cystic fibrosis (CF) is a model disease for gene therapy treatment. Doctors synthesize DNA coding for the correct version of the protein CFTR in a test tube and deliver it to patients using a virus. The new CFTR protein helps alleviate symptoms in CF patients.
83
What is **bioinformatics**?
Exploration of biological systems through the investigation of macromolecular structures using informatics techniques from mathematics, computer science, statistics, and engineering.
## Footnote
Scientists specializing in bioinformatics are called bioinformaticians.
84
What is **bioinformatics** research used for?
* Finding complex patterns in large datasets resulting from huge biological systems.
* Examining biological processes that emerge over time.
* Studying extremely small biological processes.
## Footnote
Some examples of research areas where bioinformatics can be applied:
* Evolutionary biology
* Oncology
* Gene expression
* Protein expression
85
What is **metagenomics**?
The collection of genomic data from a mixed group of organisms.
86
What is **pangenomics** used for?
To compare the genomes of several closely related organisms and study evolutionary relationships.
87
What is **proteomics**?
The study of all of the proteins produced in an organism or biological system.
## Footnote
Some examples of proteomics research:
* Finding out where, in a cell, a protein is located.
* Determining which proteins interact with which other proteins.
* Exploring the mechanics of protein-protein interactions.
* Finding out which proteins participate in which biological processes.
* Examining and predicting protein structures.
88
What is **genetic engineering**?
The process by which scientists **modify the genome** of an organism.
89
What important **applications** are associated with Genetic Engineering?
* Medicine
* Agriculture
* Commercial uses
90
When did **modern genetic engineering** start?
In 1973.
## Footnote
This was the groundbreaking work of scientists Herbert Boyer and Stanley Cohen.
91
What is **Recombinant DNA**?
A type of DNA that contains genes or sequences from **two or more species**.
92
What is a **transgenic organism**?
An organism that contains DNA from a **different species**.
## Footnote
Examples:
* BT cotton, is genetically engineered to contain genes from Bacillus thuringiensis.
* Golden Rice
93
What are **GMOs**?
Organisms that have had their DNA altered by humans.
## Footnote
Genetically Modified Organisms.
94
How is genetic engineering used in **medicine**?
* Producing human insulin in bacteria.
* Creating medical products like Interferon, human growth hormone, tissue plasminogen activator.
95
What are **glow organisms**?
Organisms engineered to express the jellyfish protein, green fluorescent protein (GFP).
## Footnote
Examples:
* GloFish
* Glowing mice
* Glowing Rabbits
96
Why is genetic engineering **controversial**?
Due to ethical and environmental concerns of altering the DNA of organisms.
97
What is a **host organism** in genetic engineering?
The organism that is **modified** in a genetic engineering experiment.
98
What is a **vector** in genetic engineering?
The **vehicle used to transfer genetic material** into a host organism.
99
What is the **purpose** of genetic engineering?
To create organisms with **desired traits** for human needs.
100
Identify characteristics of bacteria useful for genetic engineering.
Presence of **plasmids** separate from main DNA.
## Footnote
Plasmids serve as a vector for transporting genes into a host bacterial cell.
101
What is a **karyotype**?
A picture of a person's chromosomes, used to examine the number and structure of chromosomes.
## Footnote
Karyotyping tests involve creating a karyotype.
102
What is the **purpose** of a karyotype?
Used to **detect chromosome abnormalities**, such as damaged chromosomes, or having too many or too few chromosomes.
103
What is the **procedure** for conducting a karyotype?
Cells from the patient are grown in a culture and monitored for cell division. The cells are arrested in metaphase, stained, and then spread on a slide for microscopic examination.
## Footnote
A karyotype can be done during pregnancy to assess the risk of genetic disorders like Down syndrome. It is called a prenatal karyotype.
104
What conditions can be diagnosed using karyotypes?
* Down syndrome
* Chronic myeloid leukemia (CML)
## Footnote
Down syndrome is caused by an extra chromosome number 21.
Chronic myeloid leukemia (CML) involves a translocation between chromosome 9 and 22.
