21 - Manipulating Genomes Flashcards
What type of people have identical DNA?
Identical twins
What makes up an organism’s genome?
All the genetic material it contains (i.e. in eukaryotes mitochondrial DNA would be included)
What is an exon?
Region of DNA which is expressed and codes for a protein
What is an intron?
Sections of DNA which don’t code for amino acids. Introns are removed during protein synthesis.
What is satellite DNA?
Short DNA sequences which are repeated many times
What is minisatellite DNA?
A region where 20-50 base pairs is repeated 50 to several hundred times
What is another name for minisatellite regions?
Variable Number Tandem Repeats (VNTR)
In what 3 parts of DNA are satellite regions found?
Introns, centromeres, telomeres
What is microsatellite DNA?
A region of 2-4 bases repeated 5-15 times
Is satellite DNA coding or non-coding?
Non-coding
What is another name for microsatellite regions?
Short Tandem Repeats (STR)
Satellite regions always appear in the same position on what?
Chromosomes
What is DNA profiling?
Producing an image of the patterns in the DNA of an individual
What are the 5 main stages of DNA profiling?
- Extracting the DNA 2. Digesting the sample 3. Seperating the DNA fragments 4. Hybridisation 5. Seeing the evidence
Roughly what percentage of human DNA is introns?
98%
What are telomeres?
Regions of DNA at the end of chromosomes which do not code for making proteins, and which shorten each time replication occurs
What are centromeres?
The part of a chromosome that links sister chromatids during mitosis
Why does the number of STRs an individual has vary?
Lengths are inherited
What regions of DNA are examined during DNA profiling?
Satellite DNA
What technique is used to create a larger DNA sample for profiling?
PCR (Polymerase Chain Reaction)
What occurs during the extraction stage of DNA profiling?
DNA is extracted from a tissue sample
What are 2 samples which could be used to provide DNA for profiling?
- Cheek swab 2. Blood sample
What does the cutting in DNA profiling?
Special enzymes called restriction endonucleases
What is the site where a restriction endonuclease cuts a sample called?
Restriction site
How many cuts does each restriction endonuclease make?
2- one through each strand of the DNA helix
Why are a mixture of different restriction endonucleases used during DNA profiling?
To produce a mixture of different intact satellite regions
What happens in the second stage of DNA profiling?
Restriction endonucleases are used to cut up the sample
What technique is used to separate DNA fragments in DNA profiling?
Electrophoresis
What technique is used to transport the DNA from the electrophoresis gel to the nylon membrane?
Southern Blotting
What is a DNA probe?
A short, single-stranded section of DNA that has a label attached to make it easily identifiable, and which attaches to a complementary DNA sequence under particular pH or temperature conditions
What do DNA probes identify?
Microsatellite regions
What happens in the hybridisation stage of DNA profiling?
DNA probes attach to complementary DNA fragments, and excess probes are then washed off
What 2 types of label are generally added to DNA probes?
Radioactive and fluorescent tags
Are radioactive or fluorescent DNA tags more commonly used today?
Fluorescent
What does the final result of DNA profiling look like?
A pattern of bars which represents an individual’s DNA profile
How are radioactive DNA probes identified?
The membrane is x-rayed
How are fluorescent DNA probes identified?
The membrane is placed under a UV light so that they glow
What are the 3 steps of PCR?
- Separating the strands 2. Annealing primers 3. DNA synthesis
How does electrophoresis work?
The gel box has an electric field with a positive and negative end. DNA is negatively charged so it migrates to the positive end.
How is fragment size identified using electrophoresis?
It is easier for smaller molecules to move through the mesh of the box, so these travel further
What happens in the 1st stage of PCR?
Temperature increased to 90-95⁰C for 30 seconds to denature DNA
What happens in the 2nd stage of PCR?
Temperature decreased to 50-60⁰C and primers anneal to ends of DNA
What do primers do in PCR?
Allows DNA polymerase to add bases to them to build up complementary DNA strands identical to original sequence
What enzyme is used in PCR?
Taq polymerase
What happens in the 3rd stage of PCR?
Temperature increased to 72⁰C for at least 1 minute to allow taq polymerase to add bases to primers
What are 3 uses of DNA profiling?
- Crime scene investigation 2. Paternity testing 3. Identify individuals at risk of genetic diseases
What was the original method of DNA sequencing?
Sanger sequencing
How is the DNA for sequencing generated in Sanger sequencing?
By entering DNA into a bacterial plasmid
What was one refinement of the Sanger sequencing process?
Swapping radioactive labels for fluorescent ones
What are 4 disadvantages of Sanger sequencing?
- Very expensive 2. Slow 3. Can only be done 900 base pairs at a time 4. Essentially just ‘shotgunning’
What are 4 advantages of Next Gen sequencing?
- Much cheaper 2. Much quicker 3. Can sequence whole genome at once 4. Multiple genomes can be processed at once
What 2 things sped up the human genome project?
- Development of newer, more powerful computers 2. Automation of sequencing techniques
What is a terminator base?
A modified base which stops DNA synthesis
What are terminator bases given?
Fluorescent tags
What 4 things is DNA for sequencing mixed with in the 1st step of DNA sequencing?
- Terminator bases 2. Excess of normal bases 3. Primer 4. DNA polymerase
What are the 4 ingredients placed into in the second stage of DNA sequencing?
A thermal cycler