14. DNA Manipulation Flashcards
What does DNA manipulation refer to?
Altering an organism’s DNA by either adding new DNA or editing existing DNA
Genetic engineering refers to…?
The scientific method for the artificial manipulation of genes
What is a restriction enzyme?
- Endonuclease is a tool used by genetic engineers to cut DNA
- cuts DNA at precise sequences of 4-8 base pairs called recognition sequences. Once recognised the enzyme binds to DNA and cuts it in a fixed and predictable way
Where do restriction enzymes come from
Occur naturally in Bacteria, thought to have evolved as a defence mechanism against viruses
What is a recognition sequence?
The position where a cutting enzyme can snip its recognition sequence and is where a particular order of nucleotides occurs
Difference between blunt and sticky ends?
- Some restriction enzymes cut the two strands of a DNA molecule at points directly opposite to each other to produce blunt ends. Can be joined to other sticky ends with a complementary base sequence
- Other cutting enzymes cut one strand at a point but the second strand at a point that is not directly opposite. Known as sticky ends which are complementary
What is CRISPR
A complex comprising cas9 Endonuclease and sgRNA.
Cuts DNA at very specific sequences and can be used to edit genes
Where is CRISPR cas9 usually found
Occurs naturally in bacteria, which use it to chop the DNA of invading viruses
What two components are required for CRISPR to work
An RNA guide, sgRNA that locates and binds to the target piece of DNA and the cas9 Endonuclease that unwinds and cuts the DNA
What potential does the CRISPR cas9 technology have?
This technology has the potential to correct mutations responsible for genetic diseases, switch faulty genes off add new genes to an organism or to study the effects of specific genes
Cas9
Guided to the target site by sgRNA. Cas9 unwinds the DNA and cuts both strands at a specific point
Single guide RNA
Is a shot synthetic RNA sequence designed to guide cas9 to the site of interest (eg a faulty gene sequence). It contains a nucleotide section which is complementary to the DNA of interest
What is the pam sequence
It lies directly downstream of the target sequence on the non target DNA strand. Recognition of PAM by cas9 destabilises the DNA allowing the sgRNA to be inserted. Cas9 will not function if PAM is absent
Gene knock in (gene editing)
A new DNA sequence is inserted into the DNA break. For example allows a faulty gene sequence to be replaced with the correct sequence to restore normal gene function
Gene knock out (gene silencing)
As the cells normal repair process mend the broken DNA errors occur resulting in the insertion or deletion of a nucleotide bases. The resulting frame shift mutation changes the way the nucleotid sequence is read, either disabling gene function or producing a STOP signal. Can silence faulty genes
Why does DNA move in electrophoresis?
- DNA is negatively charged, due to their phosphate group in their sugar phosphate backbone
- DNA moves from negative terminal to positive terminal because DNA is negatively charged and is attracted to the positive charge at the positive terminal
What determines the speed at which fragments move?
The shortest DNA fragments move most quickly and are found further away from the starting point. The longest fragments move most slowly and are found closer to the starting point. Fragments of the same size move at the same rate.
What are standards
Consist of DNA fragments of known length and are used to compare the size of the sample DNA
How are DNA fragments made visible after the gel run?
The separated DNA bands must be made visible either through the use of a due or a radioactive probe.
One technique makes use of ethidium bromide which binds to major groove of DNA molecules.
When illuminated by u,tra violet light the DNA bound to etbr fluorescent pale pink
What is a probe?
- A probe is a single strand of DNA or RNA with base sequence that is complementary to the base sequence in one of the strands of the target DNA.
- Labelled with a radioactive or fluorescent marker so that the location of the probe and hence the target DNA can be seen.
Types of tag
Fluorescent dye tag: shows up as fluorescent bands when gel is exposed to ultraviolet light source
Radioactive tag: shows up as a dark band when the gel is exposed to photographic film
What might gene probes be used to search for?
- The position of a gene on a chromosome
- The presence of an allele of a specific gene associated with a genetic disease
- The genetic finger print of a person to identify them (paternity testing, forensic identification of subjects).
Southern blotting process
- The DNA of interest is extracted from living or dead organism, or DNA found at crime scene
- It is cut using restriction enzymes, leaving thousands of fragments of different sizes
- DNA is separated using gel electrophoresis
- DNA fragments transferred by blotting to membrane. DNA is made single stranded and fixed in place on the membrane.
- Filter sheet immersed in a bath with radioactive probes. When a probe finds a complementary base sequence it will join to the other strand. Hybridisation. Pairing between single stranded complementary DNA segments
- The probe, and hence the DNA of interest, is located, either by use of X-ray film in the case of a radioactively labelled probe where the radioactive probes show up as dark bands on X-ray film, or by fluorescence in the case of a probe with a fluorescent label.
What is ligation
The process by which DNA fragments using restriction enzymes can be joined.
What happens in the process of ligation
- An enzyme, DNA ligase catalyses the joint of pieces of double stranded DNA at the sugar phosphate backbone.
- joining can produce one larger piece of DNA or a circular molecule of DNA
- two pieces of DNA of different origins (two different species) can be joined together using restriction enzymes and DNA ligase to produce recombinant DNA