Flashcards in Lecture 14 Deck (25)
What are restriction endonucleases?
Enzymes produced in bacteria that typically recognize 4-8 bp DNA palindromic restriction sites; cuts can be staggered to yield single-stranded sticky ends or blunt to yield flush ends
What is a palindrome?
When read in the 5' --> 3' direction, the sequence on the "top" strand is identical to that of the "bottom" strand
How does a bacteria prevent its own DNA from being cleaved by a restriction enzyme?
It uses site-specific methylase to produced methylated DNA
What is DNA cloning?
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA.
What is a cloning vector?
A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes
What is gel electrophoresis?
In simple terms, electrophoresis is a process which enables the sorting of molecules based on size. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar or polyacrylamide. The electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel.
What is Polymerase Chain Reaction (PCR)?
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified
How does cystic fibrosis (CF) occur and how is it screened?
CF is an autosomal recessive disease that affects the pulmonary and digestive systems. In >70% of patients, a 3 base deletion in the coding region of the CFTR gene (cystic fibrosis transmembrane conductance regulator) results in a F508 mutation that prevents normal folding of the channel, leading to destruction of the protein by the proteasome. The 3 bp deletion in the CFTR gene can be detected by PCR
What is the difference between a mutation and a polymorphism?
A mutation is defined as any change in a DNA sequence away from normal. This implies there is a normal allele that is prevalent in the population and that the mutation changes this to a rare and abnormal variant. In contrast, a polymorphism is a DNA sequence variation that is common in the population. In this case no single allele is regarded as the standard sequence. Instead there are two or more equally acceptable alternatives. The arbitrary cut-off point between a mutation and a polymorphism is 1 per cent.
How does DNA profiling/fingerprinting work?
DNA profiling uses repetitive ("repeat") sequences that are highly variable, called variable number tandem repeats (VNTRs), in particular short tandem repeats (STRs). VNTR loci are very similar between closely related humans, but are so variable that unrelated individuals are extremely unlikely to have the same VNTRs
What is Deep DNA Sequencing?
Depth (coverage) in DNA sequencing refers to the number of times a nucleotide is read during the sequencing process. Deep sequencing indicates that the total number of reads is many times larger than the length of the sequence under study. Coverage is the average number of reads representing a given nucleotide in the reconstructed sequence.
Who was the co-discoverer of the DNA double helix and father of the Human Genome Project?
Dr. James Watson
How many base pairs are in the human genome?
3.2 billion base pairs
How many genes are there in the human genome?
What percentage of the genome encodes exons?
What part of the DNA differs between humans?
SNPs occur about 1/1250 bp
What are sources of DNA for prenatal testing?
Amniocentesis and chorionic villus sampling
What is Southern blotting?
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
What is Northern blotting?
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence.
What is Western blotting?
The western blot (sometimes called the protein immunoblot) is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide.
What are advantages of using recombinant proteins over animal/cadaver proteins?
1) Greater consistency of product quality
2) Higher efficiency of production
3) Lower immunogenicity than animal proteins
4) Reduced infectivity compared to tissue-derived products
5) Possible genetic engineering of "super proteins" with higher activity
What is cut by restriction enzymes?
Phosphodiester bonds between nucleotides
Where is DNA inserted in a plasmid?
The polylinker region
What feature do plasmids have to assist bacteria that have taken up the plasmid?
An antibiotic resistance gene, such as ampicillin resistance (ampr), is in a plasmid to separate bacteria that take up the plasmid and those that don't during DNA cloning