Module 6: Manipulating Genomes Flashcards
What is Electrophoresis?
A method of separating and ordering DNA fragments or proteins based on size.
Used so fragments can be identified and analysed.
Used in sequencing and DNA profiling
Electrophoresis stages
1) DNA can be amplified using PCR.
2) DNA is cut into smaller fragments by restriction enzymes.
3) The fragments are placed into wells at the end of the gel plate where the negative electrode (cathode) will be.
4) The plate is immersed in a tank containing buffer solution and an electric current is passed through the tank (1‐2 hours)
5) DNA is negatively charged (due to the phosphoryl groups of the sugar‐phosphate backbone) and so are attracted to the other end of the plate, where the positive electrode (anode) is, so the molecules diffuse along the gel to the other end.
6) The shorter fragments move further in the same period of time than the longer ones.
7) The banding pattern is invisible so the DNA must be stained with ethidium bromide and then viewed under UV light to observe the final banding pattern.
Electrophoresis (of proteins)
1) Done in the same way as DNA.
2) Sodium dodecyl sulfate (SDS) is added to proteins to give them equal negative charge
3) This means that they can be separated by molecular mass (rather than charge).
4) This can be used to analyse proteins by mass in blood to diagnose medical conditions:
•sickle cell anaemia
• diseases in which patients have higher levels of fetal haemoglobin than they should
What is DNA profiling?
DNA profiling (also called DNA fingerprinting ) is a way of identifying individuals by characteristics of their DNA. Often this is used to compare the DNA of more than one individual.
Almost all human DNA is the same or very similar (particularly the genes which code for proteins) so a lot of it would not be suitable for comparisons.
Short Tandem Repeats (STR)
Short Tandem Repeats (STRs) are loci on the genome composed 2‐10 base pairs which repeat between 5‐50 times in a row.
The number of repeats at each loci varies from person to person so we can use these to compare the DNA of different individuals.
About 10% of people will share the same number of repeats at any loci, so to produce a DNA profile, 13 STR loci are analysed.
The chance of two people sharing the same number repeats in each STR at 13 separate loci is about 1 x 10
13.
How to create a DNA profile
- DNA obtained from all people to be compared e.g. from saliva/hair.
- DNA amplified using PCR.
- DNA from all people cut into different size fragments using the same restriction enzymes ‐ DNA from different people will be different sizes because the number of repeats in the STR will vary.
- DNA fragments separated based on size using electrophoresis ‐ people to be compared are loaded into different wells.
- Banding pattern examined (small fragments move further ) and compared.
What is a PCR?
- A method of artificially amplifying (copying) DNA to get many copies of the same sample.
- Used to make enough DNA to test multiple times (crimes, genetic profiling).
What are DNA primers?
- 10‐20 bases of single stranded DNA
- Used for sequencing and PCR to bind to sections of DNA so that DNA polymerase can bind. DNA polymerase can’t bind to single strands, by adding primers creates a small section of a double stranded DNA which DNA polymerase can bind to.
What happens in a PCR?
• Small fragment of DNA to be copied is mixed with DNA nucleotides, primers Taq DNA polymerase and Mg
2+ (cofactors for polymerase) and heated to 95oC.
This step is called denaturation because the temperature breaks the H‐bonds between the complementary base pairs in the DNA to make 2 single strands of DNA.
• The temperature is cooled to 55 oC. This step is called the annealing step. At this temperature, the primers will bind (according the complementary base pairing rules) to each single strand of DNA at its 3’ end. This allows the DNA polymerase to bind to the double stranded sections.
- Temperature is increased to 72 oC ‐ elongation occurs as DNA polymerase adds DNA nucleotides to a single strand according to base pairing rules. This will eventually create a copy of the original fragment of DNA. Taq DNA polymerase comes from a thermophilic bacteria and so doesn’t denature at 95 degrees ‐ its optimum is 72oC
- This process is repeated over an over again and the number copies of the DNA fragment increases exponentially.
95oC and 72oC are high temperatures which would denature most enzymes including normal DNA polymerase from a human. Taq DNA polymerase comes from a thermophilic bacteria and so has a much higher optimum and won’t denature at these high temperatures
What are similarities and differences between PCR and DNA?
Similarities:
Both require polymerase, Both copy DNA.
Differences:
PCR- Heat separates complementary strands, however in DNA replication helicase separates complementary strand.
PCR- DNA primers needed for polymerase to join and
replication to begin, however in DNA replication DNA primers not needed for polymerase to join and replication to begin.
What are applications of PCR?
Forensic science - Small quantities of DNA found at a crime scene can be amplified so there is enough for DNA profiling.
Detecting mutations - Trying to find the specific mutations that caused a cancer can allow more specific medication to be given.
Uses of DNA profiling
• Forensic science:
> convicting criminals of crimes based on DNA left at crime scenes.
> identifying body parts in fires/plane crashes.
• Studying evolutionary relationships:
> e.g. finding common ancestors between different species – the more similar the banding pattern, the more closely related.
• Maternity/paternity testing:
> half of child’s DNA, and therefore half the STRs on a DNA profile, is from the mother and half from father.
What are the advantages and disadvantages of DNA profiling?
Advantages:
- Can identify presence of disorder
- Removes uncertainty
- Allows early treatment which may improve, life expectancy / quality of life
- Allows informed choice about having children
Disadvantages: - false, positives / negatives - only small number tests available / not available for all conditions - presence may not result in condition - confirmed presence gives stress / fear problem re, telling / testing, rest of family - discrimination by, employers / insurers ethics of termination
