For exam variant

What characterizes the secondary structure of a protein?

Helix or beta sheets

What is the quaternary structure of a protein?

The overall structure formed by multiple polypeptide chains connected by loops

What are fluorescent proteins useful for?

Biomedical applications

Where are fluorescent proteins located in the molecule?

In the geometric center

What tripeptide forms the fluorescent protein?

Ser-Tre-Gly

What type of reaction forms the fluorescent protein?

An autocatalytic reaction in the presence of O2

What is GFP in solution?

Monomeric (green fluorescence protein)

How are fusion proteins typically introduced into cells?

As DNA constructs

What is a cloning vector used for?

Expression of GFP fusion proteins in mammalian cells

What must be modified to create a fusion protein?

Stop codons

What does actin form in the cell?

Fibre-like structures providing stability

What doe you need for double labelling of protein skeleton?

Creation of a second pigment colour

What is IMAC?

Immobilized metal ion affinity chromatography

What are alternative methods to IMAC?

GST tagging and immunoprecipitation

How can histidine be replaced in protein purification?

By adding imidazole or protons

What is the purpose of gel filtration chromatography?

To purify proteins by size (large molecules elute first)

What is a limitation of tetrameric protein structures?

Increased size prevents them from passing through membranes

What is recombinant DNA?

DNA molecules formed by human intervention in the laboratory through genetic recombination of genetic material from different sources

What is the purpose of DNA cloning?

To isolate a DNA fragment and propagate it without altering the original DNA sequence for analysis, protein expression, or in vitro manipulation

What are the basic steps in DNA cloning?

1. Isolation of target DNA fragments. 2. Ligation of inserts into a cloning vector. 3. Transformation of recombinant plasmids into hosts. 4. Screening/selection of hosts with the intended plasmid.

What is a vector in genetic engineering?

A DNA molecule used to transfer a DNA fragment into a host cell, providing control elements for replication and expression

What are the most commonly used vectors for cloning?

Bacterial plasmids

What is the ORI?

Origin of replication

- needed within a plasmid to allow for replication

What does the AmpR region in a plasmid do?

It serves as a selectable marker for identifying bacteria that have taken up the plasmid

What is the function of the specific ORI -> pUC?

It is a specific origin of replication that produces many copies of the plasmid per cell

What is the role of the MCS (multiple cloning site)?

It contains many recognition sites for restriction enzymes

What is the promoter region in a plasmid?

Switches on the gene of interest once added

What is the purpose of restriction enzymes in cloning?

To create sticky ends or compatible ends for ligation of DNA fragments

What is ligation in the context of DNA cloning?

The process of sealing a DNA fragment into a vector to create a permanent insertion

What methods can be used for transformation of plasmids into bacteria?

Chemical transformation (e.g., heat shock) and electroporation (using electric shock)

How is selective pressure applied in cloning?

By using an antibiotic to kill cells that do not contain the plasmid

What is the first method for identifying bacterial clones with plasmids?

Selection via antibiotic resistance screening

What is the second method for identifying bacterial cells with recombinant plasmids?

White/blue screening, where blue colonies do not contain the insert, while white colonies do

What is the significance of creating sticky ends during restriction?

It ensures that the vector is going in the right direction for ligation

What is the role of phenol in DNA isolation?

It is an organic solvent used to isolate target DNA fragments

What does the term 'transformation' refer to in genetic engineering?

The process of introducing recombinant plasmids into host cells

What is the ideal length of a primer pair?

18-30 nucleotides

What is the target annealing temperature for primers?

60 degC

What should be the GC content of primers?

Around 50% (40-60%)

How should GC content be distributed in a primer?

Evenly distributed throughout the sequence as they have stronger bonds to break

What should the 3' end of a primer have to promote binding?

TLC (thymidine, leucine, cytosine)

Should have a C or G to promote binding

What is the requirement for primers in relation to the target sequence?

Primers must be homologous to the chosen sequence and nowhere else

In which direction should primers be stored?

5' to 3' direction

What is the role of MgCl in a PCR reaction?

It is a co-element for DNA polymerase synthesis

What acts as a template in a PCR reaction?

Genomic DNA

What types of primers are needed for PCR?

A forward and a reverse primer

What are DDNTPs (nucleotides) used for?

They are used for Sanger sequencing

Why is a buffer needed in a PCR reaction?

To provide the necessary environment for the in vitro system

What is the purpose of sterile water in PCR?

To ensure no microorganisms are present and to make up a certain volume

What does it mean when it says complete amplification

It means you need to amplify everything

What should be labeled in a set of primers?

3' to 5' ends

What is the requirement for the primers within the sequence?

There should be 1 primer at the beginning and 1 primer at the end of the sequence

What should a primer not finish with?

A

What is the effect of a balanced distribution of GC-rich and AT-rich domains in PCR primer design?

It avoids localized differences in Tm, improving priming

What should be avoided to prevent self-dimers or primer-dimers in PCR?

Intra-primer homology (more than 3 bases complementing within the primer) and inter-primer homology (forward and reverse primers having complementary sequences)

What are restriction enzymes used for?

Cutting DNA sequences

How are restriction enzymes named?

They are named after the species of bacteria from which they are derived

- EcoRI bacteria from e-coli

What conditions affect the activity of restriction enzymes?

Temperature, presence of co-factors, pH, and salt conditions

What is a palindromic sequence in the context of restriction sites?

A palindromic sequence can be read the same forward and backwards, such as at sticky ends

How does the length of a recognition site affect its frequency in a genome?

Longer recognition sites are less frequent than shorter ones, known as the lottery effect

What does RFLP stand for?

Restriction Fragment Length Polymorphism

What is the purpose of RFLP in genetics?

To distinguish individuals based on genetic differences, including variations in restriction enzyme cleavage sites

- detecting genetic variation

- genetic mapping

- disease diagnosis

- DNA fingerprinting

What are the steps involved in the RFLP experimental procedure?

Extraction of DNA, PCR amplification, restriction digest, size-separation by gel electrophoresis, and visualization

What is the significance of the study on Caulerpa taxifolia (Wiedenmann et al. 2001)?

Algae of Australian origin spread invasively through the Mediterranean Sea -> proved it came from an Aquarium strain, not Lessepsian migration -> used DNA fingerprinting

What is DNA sequencing?

The process of determining the nucleic acid sequence of DNA

What is the Sanger sequencing method?

Chain termination method - determining the nucleotide sequence of DNA

What is a key feature of next-generation sequencing (NGS)?

NGS allows the entire genome to be sequenced at once ('massively parallel' sequencing) in an automated process

What is the role of dideoxynucleotides in Sanger sequencing?

They terminate the DNA chain, preventing further nucleotide addition

How are different bases identified in Sanger sequencing?

By using specific fluorescent dyes that correspond to each dideoxynucleotide

What is the purpose of gel electrophoresis in DNA sequencing?

To separate DNA fragments by length for analysis

Which end does deoxyribose terminate?

3' end

What does the BLAST search tool do?

It is used to identify sequences by comparing them against a database

What is a phylogenetic tree?

A diagram that shows the evolutionary relationships among various biological species based on genetic data

What type of symbionts are thought to increase thermal tolerance in corals?

Algal symbionts of the type Symbiodinium ITS2-type D

What is the purpose of using spacer regions in RNA for identifying algae?

Spacer regions are used because they mutate without affecting function, allowing for identification

What is the significance of the study on heat-tolerant coral symbionts?

It investigates the genetic differences that enhance thermal tolerance in corals

What is the role of ethidium bromide in the RFLP process?

It is used for the visualization of DNA fragments after electrophoresis.

What is the main challenge in identifying symbionts visually?

They cannot be visually distinguished from one another

What is the outcome of the PCR amplification of the ITS2 region (Berkelmans and Oppen, 2006)?

It allows for the analysis of specific genetic markers in symbionts of heat-tolerant corals

What is the primary purpose of PCR?

To amplify a DNA fragment from a DNA template.

What enzyme is crucial for the PCR process?

DNA polymerase -> synthesises a new strand of DNA

What are the three main steps of PCR?

Denaturing, primer annealing, and extension.

What occurs during the denaturing step of PCR?

The two strands of DNA are separated by increasing the temperature to break hydrogen bonds.

What happens during the primer annealing step?

The temperature is lowered to allow primers to bind to the template DNA.

What is the result of the extension step in PCR?

DNA polymerase synthesizes new DNA strands complementary to the template.

What is meant by 'exponential amplification' in PCR?

Each cycle of PCR doubles the amount of DNA, leading to rapid increases in DNA quantity.

What is the purpose of electrophoresis after PCR?

To visualize the amplified DNA fragments.

What materials are necessary for conducting PCR?

Gloves, ice, pipettes, microtubes, PCR tubes, and a PCR instrument.

Why is contamination control important in PCR?

To prevent the amplification of incorrect DNA sequences.

What is Taq polymerase and why is it used in PCR?

A thermostable enzyme from Thermus aquaticus, ideal for high-temperature PCR cycles.

What is Pfu polymerase known for?

Its proofreading activity, which increases accuracy in DNA synthesis.

What role does Mg2+ play in PCR?

It acts as a cofactor for DNA polymerase, essential for enzyme activity.

What are dNTPs in the context of PCR?

Deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) are the building blocks for new DNA strands.

What is the significance of using both forward and reverse primers in PCR?

They ensure that both strands of DNA are amplified by providing complementary binding sites.

What is a DNA template in PCR?

The original DNA source from which the target sequence is amplified.

What types of DNA can be used as a template in PCR?

Genomic DNA, cDNA, plasmid DNA, and libraries of molecules.

How does PCR contribute to medical diagnostics?

It allows for the identification of pathogens by amplifying their DNA.

What is the role of the thermal block in a PCR instrument?

It facilitates the cycling of different temperatures required for PCR steps.

What type of bond connects the pentose sugar to the nitrogenous base in a nucleotide?

An N-glycosidic bond.

What type of bond connects the pentose sugar to the phosphate group?

An ester bond.