Human manipulation of DNA Flashcards
Chapter 1.#
What does DNA extraction consist of?
Breaking up cells and separating the nuclei through centrifuging techniques, removing the nuclear membrane with chemicals and removing the histones from DNA to isolate it.
- Obtain tissue/sample
- Centrifuge (spins) the cells at high speeds to separate the nucleus from the other smaller organelles
- Add detergents to physically break down the nuclear membrane, exposing the chromosomes
- The DNA then has to be isolated from the histone proteins
What is DNA extraction?
DNA can be transferred between species since genetic code is universal. Thus, genetic material can be manipulated .
What is a gene probe?
A segment of RNA or single-stranded DNA containing a base sequence that is complementary to part of the desired gene. This is radioactively or fluorescently labelled with a dye to allow for it to be visible.
What are restriction enzymes?
Enzymes that cut DNA (sugar phosphate backbone) at specific sites. The sequence of bases recognised by a restriction enzyme is called the restriction site and usually consists of 4-6 nucleotides.
When a restriction enzyme cuts directly across, it leaves…
…blunt ends.
When a restriction enzyme cuts diagonally across, it leaves…
…sticky ends with exposed bases.
What is Polymerase Chain Reaction (PCR)?
It is used to artificially amplify DNA samples, where the process of DNA replication is carried out artificially in a laboratory.
Describe the process of PCR.
- Denaturing: Required DNA is added to a test tube and heated to separate the two complementary strands (hydrogen bonds are weaker than the sugar-phosphate backbone of DNA)
- DNA polymerase, DNA primers (stops DNA from re-combining) and free-floating nucleotides are added
- Annealing: DNA is cooled to allow binding of free nucleotides to the exposed bases
- Extension: temperature is raised again to enable the nucleotides to bind with the exposed DNA bases, and the DNA polymerase joins them into a DNA strand
What are the two types of electrophoresis?
Gel and capillary.
What is gel electrophoresis?
A technique used to separate proteins or fragments of DNA according to their size and charge. The smaller the fragment is, the quicker it moves towards the positive charge since it has less resistance to the gel.
What is capillary electrophoresis?
It has multiple applications for DNA profiling and DNA sequencing, having mostly replaced the gel variant as it is more easily automated and faster.
What are the general principles of capillary electrophoresis?
- DNA fragments move from the negative electrode to the positive electrode
- Shorter DNA fragments move faster and further through the tube than longer ones
What is DNA sequencing?
Determines the sequence of DNA bases in a specific gene segment of DNA or the entire genome of an organism
Describe the process of gel electrophoresis.
- DNA fragments are placed at one end of a flat, rectangular gel
- Electrodes (positive and negative) are attached to the gel
- An electric current is passed through the gel
- The phosphate groups on a DNA molecule are negatively charged, meaning they move towards the positive electrode. Smaller fragments move faster than longer fragments.
- The pattern on the electrophoresis gel is transferred to a nylon sheet
What is DNA profiling? (and its applications)
A process that identifies variations in base sequences. Application examples include forensic science, paternity testing, identifying remains, medical uses, identification of seed stocks and to detect food fraud.
What is DNA profiling reliant on?
Variations in the introns that comprise ‘junk repeats’, with the number of length of these being unique to every individual. The two main categories of repeating sequences are referred to as Variable Number Tandem Repeats (VNTR) and Short Tandem Repeats (STR).
Describe the process of DNA profiling.
- Samples of DNA can be broken up into fragments using restriction enzymes; these cut at specific restriction sites.
- Every person’s DNA is different, therefore, the length of resulting fragments will also be different between people
- These different length fragments are called Restriction Fragment Length Polymorphs (RFLPs)
- DNA segments are thus separated according to size using electrophoresis
How are electropherograms interpreted?
- Computer software produces a graph of the data received from the DNA sequencer
- Each peak of the electropherogram represents one of the four DNA nucleotides
- The height of each peak represents the amount of light absorbed and emitted by the fluorescent molecule attached to the ddNTP at the ends of a DNA fragment. The order of peaks represents the nucleotide sequence of the target DNA molecule
List examples of ethical issues associated with collecting and storing genetic information.
- Are laws up-to-date?
- Privacy of consumers’ genetic information
- Surreptitious DNA testing (testing without knowledge or consent) is another privacy concern
- What if business/government servers are hacked and this information is leaked?
- Individuals could be exposed to prejudice based on their genetic profile
List examples of economic factors associated with collecting and storing genetic information.
- The overall costs associated with the collection of genetic profiles from all citizens
- This could deter people from committing crimes, and could thus mitigate the burden on the legal system where cases are backed up for long periods of time
- The creation of new jobs to collect, process, monitor and protect genetic information; job opportunities arise
List examples of ethical issues associated with collecting and storing genetic information.
- What if information is misused to ethically profile a particular group of people?
- Potential enhancement of understanding in regard to increased risks of developing specific genetic diseases for certain races/ethnicities
- Culturally based hesitation of genetic testing causes underrepresentation in genetic databases
