DNA Profiling Flashcards
Genome definition.
The genome of an organism is all of the genetic material it contains.
Genes definition.
Regions of DNA on chromosomes that code for polypeptides.
Introns definition.
Larger, non-coding regions of DNA in between the genes.
- Removed after transcription.
What are tandem repeats and variable number tandem repeats?
- Regions within the introns that are repetitive sequences of DNA that do not code for proteins.
- A variable number tandem repeat is a location in a genome where a short nucleotide is organised asa tandem repeat. Can be found on many chromosomes.
Why do the number of repeats of each mini or microsatelites (variable number tandem repeats or smaller repeated sequences) vary between individuals?
As we inherit different lengths of repeats from each parent (unless identical twins).
The more closely related you are to someone, the more likely you to have similar patterns.
Stages of DNA profiling.
- Extract DNA (from saliva, hair,semen).
- Cut DNA using restriction enzymes, which recognise palindromic sequences.
- Separate the DNA fragments using electrophoresis.
- The same is then done with the individual being compared.
- DNA must be visualised.
Restriction enzymes come from bacteria, why is that useful to the bacteria.
They can be used to defend bacterial cells from viral attacks. If the bacterium can produce restriction enzymes, it can cut up the viral DNA, making it non-functional.
How does electrophoresis work?
3) SEPARATION OF THE DNA FRAGMENTS: GEL ELECTROPHORESIS.
- Fragments need to be separated in order to look for pattens that can then be analysed.
How it works:
1- DNA fragments are put into a “well” in block of gel, along with loading dye which makes the DNA visible.
2- This is placed in an alkaline buffer solution, which controls the pH and also helps to carry the charge across the gel.
3-The gel then has an electric current passed through it.
4-The DNA fragments move towards the positive end (because the phosphate groups in the DNA give it a negative charge)
4-The smaller the fragments, the further it moves (less resistance by the gel)
5- It is important to run the gel for a sufficient amount of time, to allow all of the DNA fragments time to separate out.
How is the DNA made single stranded before analysis?
A nylon membrane is placed on top of the gel, with absorbent paper on top. This draws the top strands of DNA onto the nylon. This is called southern blotting.
What is southern blotting designed to do?
Designed to locate a particular sequence of DNA within a complex mixture. Could be used to locate particular gene within an entire genome.
DNA strands fixed in place onto nylon membrane but either UV light or heat at 85 degrees Celsius.
Fourth stage of DNA profiling.
Hybridisation
- DNA probes labelled with a fluorescent marker or radioactively.
- Probes added to DNA fragments and bind by annealing.
- Used to locate genes/ identify absence/presence of allele.
- Use x-ray or UV light for evidence.
Why do we not just use 1 STR for DNA profiling?
- There is a chance that 2 people could have the same pattern for one particular STR in their DNA.
What is PCR and what does it do?
Polymerase Chain Reaction.
- Amplifies the DNA sample - make multiple copies for it for analysis.
Process of PCR.
1- Denaturing phase:
- The temperature inside the machine is 90 degrees Celsius for 20 secs.
- This breaks the hydrogen bonds between the 2 DNA strands, separating them.
2- Annealing phase:
- Temperature is decreased to 60 degrees Celsius.
- Primers anneal to both ends of the single DNA strands.
3-Extension phase:
- Temperature increased to 70 degrees Celsius for one min.
- This is the optimum temperature for the chosen DNA polymerase enzyme to work. This adds base to the primers, extending the complimentary strands.
- This results in double-stranded DNA genetically identical to the original sample.
What is the polymerase used in the extension phase called and how is it adapted?
Polymerase used is called “Taq polymerase” and it comes from thermophilic hot-spring bacteria. This means that it is not denatured by the high temperatures needed inside the PCR machine.
Why might you not get as many copies as expected for PCR?
- There may not be enough primers present.
- The primers may not attach to all the DNA strands.
- There may be insufficient nucleotides available.
- When the 2 DNA strands are separated, they may just re-join to each other instead of the primers.
What is the difference between a “probe” and a “primer”?
A probe is 100-1000 nucleotides long and is used to locate a particular base sequence.
A primer is 15-25 nucleotides long and is used to start the replication of the DNA strands during PCR technique.
