C25 - Gene Technologies Flashcards
What does PCR stand for?
Polymerase chain reaction
What is PCR
The polymerase chain reaction is the DNA amplification method used to increase the amount of DNA available.
It’s an in vitro method for copying and amplifying sections of DNA and is carried out in a thermocycler.
What molecules are placed in the thermocycler during PCR?
The DNA fragments being copied
Free phosphorylate nucleotides (which will be bonded together to form copies of the DNA fragment)
Taq DNA polymerase (which binds the free nucleotides together)
Primers
Why is Taq DNA polymerase used in PCR instead of human DNA polymerase?
Taq DNA polymerase is obtained from a thermophilic bacterium (thermus aquaticus).
This form of polymerase is tolerant to heat so doesn’t denature during PCR temperature cycling.
What are primers?
Short sequences of DNA, complementary to one end of a DNA fragment (used in PCR).
They provide the starting sequence for DNA polymerase to begin the copying process.
What are the (3) stages of the polymerase chain reaction (PCR)?
1) At 95°C, the DNA strands of the fragments are separated. There is enough kinetic energy to break the hydrogen bonds between DNA strands.
2) The thermocycler mixture is cooled to 55°C. This allows primers to form hydrogen bonds to complementary bases at the end of each DNA strand.
3) The thermocycler is raised to 72°C (optimum temp for the Taq DNA polymerase). The polymerase joins free complementary nucleotides to each of the separated DNA strands, beginning at the primers.
What temperatures are used in (the 3 stages of) the PCR?
95°C
55°C
72°C
What is gel electrophoresis?
A DNA analysis technique which separates DNA by size, by an electric current.
This allows the separation of fragments that differ by a single base.
What are the steps in gel electrophoresis?
1) An electrophoresis gel plate consists of a agarose (gel) covered in buffer solution. Electrodes are attached at both ends, which enables an electric current to be passed through the gel.
2) The DNA sample is cut into fragments using restriction endonuclease enzymes.
3) The DNA fragments are placed in wells in the gel at the end closest to the negative electrode.
4) When a current is applied, DNA moves towards the positive electrode (anode) because of its negatively charged (phosphate) groups.
5) Longer fragments don’t move as far because they interact with the gel more than short fragments.
Shorter fragments will therefore be closer to the anode at the end of the procedure.
6) The fragments appear as bands which can be visualised using UV light and a fluorescent DNA dye.
7) DNA fragments can be extracted from the agarose gel for further analysis.
What does VNTR stand for?
Variable number tandem repeats
What are VNTRs (variable number tandem repeats)?
Patterns of repeated nucleotides adjacent to each other in a DNA sequence (e.g. CAATT CAATT CAATT).
Changes in VNTR nucleotide sequences alter the positions at which restriction endonucleases cut DNA.
How do VNTRs differ between people?
Variable number tandem repeats vary between different people - the probability of 2 unrelated people having the same VNTR pattern is very low.
Therefore nucleotide sequences of VNTRs can be used as genetic fingerprints for analysis.
What does SNP stand for?
Single nucleotide polymorphism
What are SNPs (single nucleotide polymorphisms)?
Sequences of DNA that can vary between people by a single nucleotide.
They can occur in genes, but are more common in non-coding DNA. When SNPs occur in genes, they’re referred to as alleles and are caused by substitution mutations.
Some can indicate a persons susceptibility to disease and how they respond to certain drugs.
What’s a haplotype?
A set of genes inherited together from one parent.
As a result of the founder effect, people with the same ancestral origins tend to share SNPs and have very similar haplotypes.
What steps are involved in the genetic engineering of DNA from different species?
1) Obtaining the required gene
2) Placing the gene in a vector (a structure that carries the gene into the recipient cell)
3) Transporting the gene into the recipient cell
What 2 methods of gene modification are used on bacteria?
1) Cutting our the gene using restriction enzymes
2) Producing the gene from an mRNA template
How are restriction enzymes used to extract a gene?
There are over 50+ different restriction enzymes, each of which cuts DNA at specific base sequences called ‘recognition sites’.
Many enzymes produce a staggered cut which creates 2 short sequences of exposed, unpaired bases known as sticky ends. These are often palindromic.
Plasmids can be used as vectors.
The required gene and plasmids are cut with the same restriction enzyme, creating 2 sets of sticky ends with complementary base pairings.
The plasmid and recombinant gene are annealed and DNA ligase is used to join the sticky ends on the gene and plasmid.
The gene becomes integrated into the plasmid vector and the resultant DNA (containing sections from 2 organisms) is known as a recombinant plasmid.
Where are restriction enzymes found?
In bacteria.
50+ different enzymes are available for engineering, each of which cut DNA at specific base sequences known as recognition sites.
What’s a recognition site?
A specific sequence of bases in the DNA, recognised and cut by restriction enzymes.
How is DNA cut by restriction enzymes?
They identify recognition sites and produce a staggered cut.
This creates 2 short sequences of exposed, unpaired bases known as sticky ends.
The enzyme recognition sequences are often palindromic.
