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DNA libraries & generating cDNA | Flashcards

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1
Q

What are proteins made up of?

A

Chains of amino acids

Proteins are fundamental molecules in living organisms.

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2
Q

What is the base material for protein synthesis?

A

DNA (Deoxyribonucleic Acid)

DNA serves as the template from which proteins are created.

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3
Q

What is the first step in protein synthesis?

A

Transcription of DNA to produce messenger RNA (mRNA)

This process is essential for carrying genetic information to ribosomes.

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4
Q

What is the function of mRNA?

A

Carries genetic information from DNA to ribosomes

mRNA is crucial for protein synthesis.

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5
Q

What is the reverse process in molecular biology?

A

Determining DNA sequence from a protein

This process is important for research and clinical diagnostics.

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6
Q

Define a DNA library.

A

A collection of DNA sequences that researchers can query

DNA libraries help find genetic sequences corresponding to specific proteins.

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7
Q

What is the starting point for creating a DNA library?

A

The amino acid sequence of the target protein

This sequence is linked to the mRNA sequence.

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8
Q

What enzyme is used to convert mRNA back into cDNA?

A

Reverse transcriptase

cDNA stands for complementary DNA.

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9
Q

What is the difference between regular cellular DNA and cDNA?

A

Regular DNA is double-stranded; cDNA is initially single-stranded

cDNA complements the mRNA sequence.

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10
Q

What enzyme is utilized to convert single-stranded cDNA into double-stranded DNA?

A

DNA polymerase

This step generates stable genetic material for further manipulation.

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11
Q

What must double-stranded DNA be inserted into for sequencing?

A

A cloning vector such as a plasmid or virus cloning vector

Cloning vectors allow for the propagation of inserted DNA.

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12
Q

What is the purpose of amplification in DNA sequencing?

A

To produce multiple copies of double-stranded DNA

This is achieved by replicating the DNA within a host organism.

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13
Q

What happens to the sequences obtained after sequencing the DNA?

A

They are entered into a comprehensive database

This facilitates quick retrieval for future research or clinical inquiries.

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14
Q

The entire process of synthesizing proteins from DNA illustrates the complexity of what field?

A

Molecular biology techniques

It highlights the vital role of DNA libraries in advancing scientific knowledge.

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15
Q

What is the main purpose of cDNA in molecular biology?

A

Facilitates the study of gene expression, functional genomics, and isolation of coding sequences

cDNA is synthesized from mRNA templates.

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16
Q

What is the process of synthesizing cDNA from mRNA called?

A

Reverse transcription

This involves using reverse transcriptase to create cDNA from mRNA.

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17
Q

List the steps involved in the generation of cDNA from mRNA.

A
  • Isolation: mRNA is extracted
  • Priming: A primer is annealed
  • Reverse Transcription: cDNA is synthesized
  • RNA Degradation: RNA is hydrolyzed

These steps are crucial for creating cDNA from mRNA templates.

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18
Q

True or false: cDNA contains non-expressed sequences of chromosomes.

A

FALSE

cDNA selectively captures expressed genes.

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19
Q

What are the advantages of using cDNA?

A
  • Selective: Captures expressed genes
  • Full-Length Copies: Preserves entire mRNA sequences

These advantages make cDNA valuable for gene expression studies.

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20
Q

What enzyme is used to synthesize cDNA from mRNA?

A

Reverse transcriptase

This enzyme is essential for the reverse transcription process.

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21
Q

Can cDNA be used for gene therapy or constructing transgenic animals?

A

Yes

cDNA can be utilized in various applications, including gene therapy.

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22
Q

What type of sequences does a genomic library contain?

A

Contains non-expressed sequences of chromosomes

This contrasts with cDNA libraries, which only include expressed genes.

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23
Q

What is a key difference between cloned genes in a genomic library and a cDNA library?

A
  • Genomic library: Cloned genes contain introns
  • cDNA library: Cloned genes are complete sequences

This difference is significant for gene expression studies.

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24
Q

Fill in the blank: The process of synthesizing cDNA from mRNA involves the enzyme _______.

A

Reverse transcriptase

This enzyme is crucial for the reverse transcription process.

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25
What is the first step in the **expression of cloned genes**?
Isolation of mRNA ## Footnote Extract mRNA from the source organism or tissue using techniques like RNA extraction kits.
26
What enzyme is used in **reverse transcription** to convert mRNA into cDNA?
Reverse transcriptase ## Footnote This enzyme is crucial for synthesizing complementary DNA from mRNA.
27
What process is used to amplify cDNA for cloning?
Polymerase chain reaction (PCR) ## Footnote PCR generates sufficient amounts of cDNA for further cloning.
28
What is introduced into a suitable **cloning vector** during gene expression?
cDNA ## Footnote Cloning vectors can include plasmids or viral vectors, using restriction enzymes and ligases.
29
What is the purpose of **transformation of host cells** in gene expression?
Introduction of the recombinant plasmid carrying the cDNA ## Footnote Typically, host cells are bacteria like Escherichia coli or other expression systems.
30
What is used to induce **gene expression** in host cells?
Specific promoters or inducible systems ## Footnote These elements control the expression of the cloned gene.
31
What is the first step in the **Polymerase Chain Reaction (PCR)** process?
Denaturation ## Footnote DNA strands are separated at high temperature during this step.
32
What occurs during the **annealing** step of PCR?
Primers bind to complementary sequences on each DNA strand ## Footnote This step is crucial for initiating DNA synthesis.
33
What is the role of **DNA polymerase** in PCR?
Synthesizes new strands from the primers ## Footnote This enzyme is essential for DNA strand extension.
34
What are the components required for PCR? List them.
* DNA Template * Primers * DNA Polymerase * Nucleotides ## Footnote These components are necessary for the amplification of DNA.
35
What are some **applications** of PCR?
* Genetic Research * Diagnosis * Forensic analysis ## Footnote PCR is widely used for amplifying specific genes or DNA regions.
36
True or false: PCR is a rapid method for amplifying DNA.
TRUE ## Footnote PCR allows for quick amplification of specific DNA sequences.
37
What significant impact has PCR had on **molecular biology**?
Revolutionized DNA amplification ## Footnote PCR has contributed to advancements in various scientific and medical fields.
38
What is the **basic principle** of **Gel Electrophoresis**?
* Separation of proteins, DNA, or RNA based on size and/or charge * Electric field causes negatively charged substances to move * Bottom: (+) charged cathode and top: (-) charged anode * Porous gel traps larger substances while smaller ones move faster ## Footnote Gel electrophoresis is a technique used to separate biomolecules for analysis.
39
How does **DNA** behave in gel electrophoresis?
* Carries overall (-) charge due to phosphate backbone * Migrates toward (+) charged cathode in electric field * Fragments visualized by fluorescent dye (ethidium bromide) ## Footnote Ethidium bromide intercalates between DNA strands and fluoresces under UV light.
40
What is the purpose of **Southern Blotting**?
* Identifies specific DNA sequences ## Footnote The procedure includes DNA electrophoresis, transfer to membrane, hybridization with labeled probes, and detection.
41
What is the procedure involved in **Southern Blotting**?
* DNA electrophoresis * Transfer to membrane * Hybridization with labeled probes * Detection ## Footnote This method is used for analyzing genetic variations or locating specific genes.
42
What is the purpose of **Northern Blotting**?
* Identifies specific RNA molecules ## Footnote Similar to Southern Blotting, it involves RNA electrophoresis, transfer to membrane, hybridization with labeled probes, and detection.
43
What is the procedure involved in **Northern Blotting**?
* RNA electrophoresis * Transfer to membrane * Hybridization with labeled probes * Detection ## Footnote This method is used for analyzing mRNA levels for gene expression studies.
44
What is the **Western Blot** used for?
* Identifies specific proteins ## Footnote The process involves protein extraction, electrophoresis, transfer to membrane, and detection.
45
List the three types of blotting techniques mentioned.
* Southern Blot * Northern Blot * Western Blot ## Footnote Each technique is used for different types of biomolecules: DNA, RNA, and proteins respectively.
46
What is the **purpose** of **Western Blotting**?
Identifies specific proteins ## Footnote Western Blotting involves protein electrophoresis, transfer to membrane, antibody binding, and detection.
47
List the **applications** of **Western Blotting**.
* Analyzing protein levels * Modifications * Interactions ## Footnote Western Blotting is used in various fields including research and diagnostics.
48
What is the **purpose** of **Enzyme-Linked Immunosorbent Assay (ELISA)**?
Detects and quantifies specific proteins or antibodies ## Footnote ELISA is widely used for sensitive and rapid protein detection.
49
List the **types** of **ELISA**.
* Direct ELISA * Indirect ELISA * Sandwich ELISA ## Footnote Each type has a different methodology for detecting the target analyte.
50
What are the **applications** of **ELISA**?
* Diagnostics for infectious agents * Biomarker detection * Research ## Footnote ELISA is significant for its sensitivity and rapid results.
51
Fill in the blank: The **procedure** for **Western Blotting** includes protein electrophoresis, transfer to membrane, ________, and detection.
antibody binding ## Footnote This process is crucial for identifying specific proteins.
52
Fill in the blank: The **procedure** for **ELISA** includes coating, blocking, binding, and ________ detection.
enzyme-driven ## Footnote This method allows for quantification of proteins or antibodies.
53
In **Sandwich ELISA**, what are the components involved?
* Antigen * Antibody * Secondary Antibody * Reporter Enzyme ## Footnote This format enhances the sensitivity of the assay.
54
What is the purpose of **DNA Sequencing**?
To determine the sequence of nucleotides in a strand of DNA ## Footnote Application: Detect specific mutations to diagnose genetic diseases.
55
What is **Sanger sequencing**?
A widely-used method for determining the nucleotide sequence of DNA ## Footnote It involves DNA synthesis with chain-terminating dideoxynucleotides and electrophoretic separation of the resulting fragments.
56
List the components required for **Sanger sequencing**.
* A radiolabeled DNA primer * DNA polymerase * All four dNTPs (dATP, dTTP, dCTP, dGTP) * Small quantity of single daNTP (e.g., ddATP) ## Footnote The resulting fragments are separated by size in a gel, and the sequence is revealed through electropherogram analysis.
57
What are **dideoxynucleotides (ddNTPs)**?
Modified nucleotides missing OH group on 3' carbon ## Footnote They are unable to create a new 5'-3' phosphodiester bond and cause chain termination when incorporated into a synthesizing DNA molecule.
58
What does **Next Generation Sequencing** enable?
High-throughput sequencing, simultaneously analyzing millions of DNA fragments ## Footnote Represents a transformative advancement in DNA sequencing technology with applications in genomics, transcriptomics, and epigenomics.
59
What is the purpose of **Karyotyping**?
Analyzes chromosome number and structure ## Footnote Crucial for diagnosing genetic conditions and understanding chromosomal abnormalities.
60
What are the steps involved in the **Karyotyping** procedure?
* Cell culturing * Harvesting * Slide preparation * Microscopic examination ## Footnote Applications include identifying genetic disorders and detecting chromosomal changes in cancer.
61
True or false: **Karyotyping** provides visual insight into an individual's chromosomal composition.
TRUE ## Footnote It is significant for diagnosing genetic conditions and understanding chromosomal abnormalities (e.g., Klinefelter syndrome, Turner syndrome).
62
What is the primary purpose of **karyotyping**?
Diagnosing genetic conditions by visualizing chromosomal abnormalities ## Footnote Karyotyping guides medical interventions and counseling.
63
What genetic condition is identified by an extra copy of **chromosome 21**?
Down Syndrome ## Footnote This condition is also referred to as trisomy 21.
64
What characterizes **Klinefelter Syndrome**?
An additional X chromosome in males (XXY) ## Footnote This condition is detectable through karyotyping.
65
What occurs in **Turner Syndrome**?
A missing X chromosome in females (X) ## Footnote This condition is evident in karyotypes.
66
What is the function of **Fluorescence In Situ Hybridization (FISH)**?
Locates and visualizes specific DNA sequences in cells ## Footnote FISH aids in detecting chromosomal abnormalities and gene localization.
67
What is the first step in the **FISH procedure**?
Probe Hybridization ## Footnote Fluorescently labeled DNA probes bind to target sequences.
68
What follows Probe Hybridization in the **FISH procedure**?
Washing ## Footnote Excess probes are washed away after hybridization.
69
What technique is used to amplify and quantify **RNA molecules**?
Reverse Transcription PCR (RT-PCR) ## Footnote RT-PCR converts RNA to complementary DNA using reverse transcriptase.
70
What is the initial step in **RT-PCR**?
Reverse transcription converts RNA to complementary DNA ## Footnote This process uses reverse transcriptase.
71
What does **Quantitative PCR (qPCR)** measure?
The abundance of a specific DNA target ## Footnote qPCR utilizes fluorescent probes for real-time monitoring during DNA amplification.
72
List three applications of **qPCR**.
* Gene expression studies * Pathogen detection * Various molecular biology applications ## Footnote qPCR offers precise and rapid quantification of nucleic acids.
73
What is the purpose of **Gene Knockout**?
Inactivates a specific gene to study its function ## Footnote Gene Knockout occurs via two main mechanisms.
74
Name the two main mechanisms of **Gene Knockout**.
* Gene Targeting * Cell or Organism Culture ## Footnote Gene Targeting introduces a disrupted or inactive copy of the gene.
75
What is the purpose of **Studying Protein Function by Comparing DNA Sequences**?
Analyzes DNA sequences to deduce protein functions ## Footnote This process involves sequence alignment, homology search, and functional annotation.
76
List the steps involved in **Studying Protein Function by Comparing DNA Sequences**.
* Sequence Alignment * Homology Search * Functional Annotation ## Footnote These steps help in associating unknown genes with known functions.
77
What are the **applications** of studying protein function through DNA sequence comparison?
* Gene Function Prediction * Drug Target Identification ## Footnote This is crucial for understanding gene roles in cellular processes and facilitates drug discovery.
78
What are the types of **Stem Cells** mentioned?
* Embryonic Stem Cells (ESCs) * Induced Pluripotent Stem Cells (iPSCs) * Mesenchymal Stem Cells (MSCs) * Hematopoietic Stem Cells (HSCS) * Neural Stem Cells (NSCs) * Tissue-Specific Progenitor Cells ## Footnote Each type has unique properties and applications in research and medicine.
79
What are **Embryonic Stem Cells (ESCs)** derived from?
Embryos ## Footnote ESCs are pluripotent but face ethical concerns.
80
What are **Induced Pluripotent Stem Cells (iPSCs)** reprogrammed from?
Somatic cells ## Footnote iPSCs are pluripotent and ethically more acceptable.
81
Where are **Hematopoietic Stem Cells (HSCS)** located?
Bone marrow and blood ## Footnote HSCS are crucial for blood cell production.
82
What is the role of **Neural Stem Cells (NSCs)**?
Differentiate into neural specialized cell types ## Footnote NSCs are present in the nervous system and are important for tissue maintenance and repair.
83
True or false: **Mesenchymal Stem Cells (MSCs)** are multipotent and used in regenerative therapies.
TRUE ## Footnote MSCs are found in tissues and hold significant potential for various applications.
84
What is the role of **recombinant DNA technology** in vaccines?
Identify protein components of viruses or microbial pathogens that elicit an antibody response ## Footnote Vaccines lead to an antibody response without an infection, protecting the host from the pathogen.
85
What is the purpose of **vaccines**?
Lead to an antibody response without an infection ## Footnote This protects the host from future infections.
86
Name an example of a **vaccine**.
* Hepatitis B virus * Herpes virus * Malaria ## Footnote These vaccines help in eliciting an immune response.
87
What is **gene therapy** used for?
Fix pathology by transferring normal gene copy into mutated tissues ## Footnote This can involve using modified viruses as gene delivery vectors.
88
What technology allows for precise targeting and cutting of genes?
CRISPR ## Footnote CRISPR is a form of gene therapy that leads to genetic modification.
89
What does **pharmacogenomics** study?
How genetic variation can influence drug response ## Footnote This aims to improve the safety and efficacy of drugs.
90
How is DNA technology used in **forensics**?
Identify specific individuals by looking at non-coding regions in the genome ## Footnote This helps in criminal investigations and paternity testing.
91
What is one application of recombinant DNA technology in **agriculture**?
Create crops resistant to insects and herbicides ## Footnote This includes the ability to delay ripening.
92
What is **phytoremediation**?
Using plants to clean the environment ## Footnote This is one application of recombinant DNA technology.
93
What is **microbial remediation**?
Using microbes to clean the environment ## Footnote This is another application of recombinant DNA technology.
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