DNA Analysis 2 Flashcards
What can gel electrophoresis be used for
What is an electropherogram
How does electrophoresis work
What are the equations for finding electrophoretic velocity and electrophoretic mobility
How can you speed up electrophoresis and what is the side effect of doing so
How is shape important in electrophoresis
What is denaturants are used in electrophoresis and why
What can non denaturing polyacrylamide gel electrophoresis be used for
What can this calibration curve be used for
Where does agarose come from
What are the characteristics of agarose
Higher % of agarose= greater separation of maecules in the gel
What are the components of equipment used in gel electrophoresis
What is CHEF
What is polyacrylamide made up of
What are the characteristics of polyacrylamide
Higher % of acrylamide = greater separation of maecules in the gel
What is common configurations
How does protein electrophoresis work
What is the Ferguson plot
What is a gradient gel
How can the size of molecules be estimated using gel electrophoresis
What are the different stains used in electrophoresis
How does ethidium bromide work and what are the issues with its use
How does sybr dye work
How does coomassie blue dye work
What is blue native PAGE and how does it work
What is hybridisation and dissociation of DNA
What are the factors affecting hybridisation /denaturation
What is the hyperchromic effect
the increase in absorbance of DNA upon denaturation
How can hybridisation be observed
Hyperchromic Effect: This effect refers to an increase in the absorption intensity (absorbance) of a molecule compared to its individual components
Hypochromic Effect: the hypochromic effect refers to a decrease in the absorption intensity of a molecule compared to its individual components
What is Tm
Melting point of DNA- when half of strand is associated with the complimentary strand and the other half is dissociated
What can cause an increase in Tm
What algorithms can be used to figure out Tm
What is a probe and target
What is the order of stability of hybrids
What is low and high stringency of hybridisation
Stringency refers to the conditions under which this hybridization occurs, affecting the specificity and stability of the formed hybrids.
Low stringency conditions:
In low stringency conditions, the temperature and salt concentration are relatively low, allowing for more relaxed hybridization conditions.
Low stringency conditions favor the formation of hybrids between nucleic acid strands with some degree of mismatch in their base sequences.
This can be useful for detecting related sequences with some degree of similarity, such as identifying homologous genes across different species or detecting gene families within a genome.
Low stringency conditions are also employed in techniques like Southern blotting, Northern blotting, and in situ hybridization.
High stringency conditions:
In high stringency conditions, the temperature and salt concentration are higher, promoting more stringent hybridization conditions.
High stringency conditions favor the formation of hybrids only between nucleic acid strands with a high degree of sequence complementarity.
This allows for more specific detection of target sequences, reducing nonspecific binding or cross-reactivity.
High stringency conditions are commonly used in techniques like polymerase chain reaction (PCR) and DNA microarray analysis, where specific detection of target sequences is critical.
What is homologous hybridisation
Homologous hybridization, also known as homology-based hybridization, is a molecular biology technique used to detect sequences in a sample that are similar or identical to a known probe sequence. In this technique, a labeled DNA or RNA probe, which is complementary to the target sequence of interest, is hybridized to the sample DNA or RNA under specific conditions
What is heterologous hybridisation
Heterologous hybridization, also known as heterology-based hybridization, is a molecular biology technique used to detect sequences in a sample that are not necessarily similar or identical to a known probe sequence. In this technique, a labeled DNA or RNA probe, which may have limited or no sequence homology with the target sequence of interest, is hybridized to the sample DNA or RNA under specific conditions.
What is a homologous sequence
What are examples of techniques that exploit hybridisation
What is blotting
What types of blotting are there
What is the process of southern blotting
What are the uses of the different types of blotting
What is colony hybridisation
How are DNA microarrays carried out
How is in situ hybridisation carried out
How is DNA typically represented
Explain the lytic cycle of a bacteriophage
What is phage plaque
(Dead bacteria)
Lawn = where bacteria has grown
What do restriction enzymes do in bacteria
Restriction enzymes, also known as restriction endonucleases, are enzymes found in bacteria that play a crucial role in the bacterial immune system against foreign DNA, such as viral DNA (bacteriophages). These enzymes recognize specific DNA sequences and cleave the DNA at or near these sequences. This process is part of the bacterial defense mechanism against foreign DNA.
The concept of “restriction of host ranges” in bacteria using restriction enzymes refers to the ability of bacteria to protect themselves from infection by bacteriophages that carry DNA sequences not recognized by the bacteria’s own restriction enzymes.
When a bacteriophage infects a bacterial cell and injects its DNA, the bacterial restriction enzymes recognize the foreign DNA of the bacteriophage as non-methylated and thus foreign. The restriction enzymes bind to the recognition sites on the foreign DNA and cleave it, rendering it non-functional.
Replication: By cleaving the foreign DNA of the bacteriophage, the bacterial restriction enzymes prevent the replication and propagation of the bacteriophage within the bacterial host. This inhibits the infection cycle of the bacteriophage and protects the bacterial cell from lysis and death caused by viral replication
What is the difference between bacteria and phages (bacteriophages)
Bacteriophages are viruses that infect bacteria and can kill bacteria
How do restriction enzymes work in a bacteriums immune system
What are nucleases
What are the products of hydrolysis of nucleic acids
How are restriction enzymes named
What are the 3 different types of restriction enzyme endonucleases
Type I Restriction Endonucleases:
Type I restriction endonucleases are large, multisubunit enzymes that recognize specific DNA sequences but cleave DNA at sites that are distant from the recognition sequence.
These enzymes require both ATP hydrolysis and S-adenosylmethionine (SAM) as cofactors for their activity.
Upon binding to the recognition sequence, Type I restriction endonucleases can translocate along the DNA molecule in a processive manner, searching for specific sequences. Once they locate a specific site, they cleave the DNA randomly at sites far away from the recognition sequence.
The cleavage sites are often several thousand base pairs away from the recognition site.
Type II Restriction Endonucleases:
Type II restriction endonucleases are the most common type and are widely used in molecular biology applications, such as DNA cloning and gene editing.
These enzymes recognize specific DNA sequences, typically palindromic sequences, and cleave the DNA within or near the recognition sequence.
Unlike Type I enzymes, Type II restriction endonucleases do not require ATP or SAM for their activity.
The cleavage sites are usually symmetrical and are often within or near the recognition sequence, resulting in blunt ends or overhangs (sticky ends) depending on the enzyme.
Type III Restriction Endonucleases:
Type III restriction endonucleases are similar to Type I enzymes in that they recognize specific DNA sequences and cleave DNA at sites that are distant from the recognition sequence.
These enzymes require ATP hydrolysis for their activity.
Like Type I enzymes, Type III restriction endonucleases can translocate along the DNA molecule in a processive manner and cleave the DNA at sites far away from the recognition sequence.
The cleavage sites are often several hundred base pairs away from the recognition site.
What is a palindrome, mirror like palindrome and an inverted repeat palindrome
What is hemimethylated DNA
What happens to unmethylated DNA in a cell
How can DNA be protected from degradation from a restriction enzyme
How do type 1 restriction modification systems work
How do type 2 restriction modification systems work
How do type 3 restriction modification systems work
What is the product of DNA restriction enzyme digestion
Forms sticky ends
Different restriction enzymes produce different sticky ends
What are ‘blunt ends’
What are isoschizomers
What are the uses of restriction enzymes
What is the role of DNA polymerase
What is the process of PCR
What is taq polymerase
How is PCR represented graphically
What is the equation that tells you the final amount of DNA produced after PCR and what does this look likegraphically
Equation - Y= (2^x)A
Y = final amount of dna produced
X= number of cycles of PCR
A= initial amount of dna in sample
How is the DNA produced from PCR analysed
What is the name of the machine used in PCR
Thermal cycler
What is the problem with taq polymerase
What is PCR used for
Quantification
Site directed mutagenesis
How does quantitative PCR work
What is the role of PCR in site directed mutagenesis
