Module 6.3 - Manipulating Genomes Flashcards
Define the Polymerase Chain Reaction
A method of artificially amplifying DNA to get many copies of the same sample
Uses of PCR
To make enough DNA to test multiple times (crimes, genetic profiling)
Identifying viral infections (can detect small amounts of viral DNA amongst host DNA - can be used to test for e.g. HIV)
Detecting mutations (DNA analysed to look for mutations that cause genetic disease - can be done in parents/embryos)
Forensic science (small quantities of DNA found at a crime scene can be amplified so that there is enough for DNA profiling)
Tissue typing (donor and recipient tissues can be ‘typed’ to reduce risk of rejection in transplants)
Detection of oncogenes/cancer genes (trying to find specific mutations that caused a cancer can allow more specific medication to be given)
Research (can amplify sources of DNA from fossils for sequencing to study evolutionary relationships - in living species genes which are switched on or off can be studied)
What are DNA primers?
10-20 bases of single stranded DNA
Uses of DNA primers
Sequencing and PCR to bind to sections of DNA so that DNA polymerase can bind as DNA polymerase can’t bind to single strands
PCR key steps and temperatures
Denaturation - 95°C
Annealing - 68°C
Elongation - 72°C
Steps of PCR
Small fragment of DNA to be copied mixed with DNA nucleotides, primers and Taq DNA polymerase (comes from a thermophilic bacteria so will not denature at 95°C, optimum is 72°C)
Heated to 95°C - denaturation
High temperature breaks H-bonds between complementary base pairs in DNA to make 2 single strands of DNA
Temperature cooled to 55°C - annealing
Primers bind to each single strand of DNA at the 3’ end, allowing DNA polymerase to bind to double stranded sections
Heated to 72°C - elongation
Taq DNA polymerase adds DNA nucleotides to single strand according to complementary base pairing rules
This will eventually create a copy of the original fragment of DNA
This process is repeated over and over again and the number of copies of the DNA fragment increases exponentially
Differences between PCR and DNA replication
PCR heat separates complementary strands, DNA replication DNA helicase and girase separate strands
PCR DNA primers needed for polymerase to join and replication to begin, DNA replication DNA primers not needed
PCR does not copy whole chromosome, DNA replication copies whole chromosome
PCR repeats immediately after one cycle done, DNA replication repeats once every cell cycle
PCR Taq DNA polymerase used, DNA replication DNA polymerase used
PCR artificial DNA replication, DNA replication is natural
Similarities between PCR and DNA replication
Copy DNA
Require polymerase
Define electrophoresis
Method of separating and ordering DNA fragments or proteins based on size
Uses of electrophoresis
Fragments can be identified and analysed
Sequencing and DNA profiling
Steps of electrophoresis of DNA
Small amounts of DNA can be amplified using PCR
DNA is cut into smaller fragments using restriction enzymes (the same restriction enzymes must be used to cut the fragments for any of the individuals involved in the identification for forensics)
The fragments are placed into the wells at the end of the gel plate where the negative electrode/cathode will be
The plate is immersed into a tank filled with buffer solution and an electric current is passed through the tank for 1-2 hrs
DNA is negatively charged (due to phosphoryl groups of 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
The shorter fragments move further in the same period of time than the longer ones
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
Steps of electrophoresis of proteins
Done int he same way as DNA
Sodium dodecyl sulphate (SDS) is added to proteins to give them equal negative charge
This means that they can be separated by molecular mass rather than charge
This can be used to analyse proteins by mass in blood to diagnose medical conditions (e.g. sickle cell anaemia, diseases in which patients have higher levels of foetal haemoglobin than they should)
Define DNA profiling
Way of identifying individuals by characteristics of their DNA
Often used to compare DNA of more than one individual
What are Short Tandem Repeats (STRs)?
Loci on the genome composed of 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
How do we find out the number of STRs a person has at each location?
Electrophoresis
More repeats in STRs = larger DNA fragment = doesn’t move as far in electrophoresis
Steps of creating a DNA profile
DNA obtained for all people to be compared (elf, 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 as 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
Uses of DNA profiling
Forensic science (convicting criminals of crimes based on DNA left at crime scenes, identifying body parts in fires/plane crashes)
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)
Studying evolutionary relationships (finding common ancestors between different species, the more similar the banding pattern the more closely related)
Analysis of disease/genetic screening (some diseases are caused by STRs which repeat too many times e.g. Huntington’s)
Advantages of genetic screening
Can identify presence of a disorder Removes uncertainty Allows early treatment May improve life expectancy/quality of life Allows informed choice about having children Allows IVF and embryo screening Allows foetal testing and termination Choice of adoption
Disadvantages of genetic screening
False positives/negatives
Only small number of tests available, not available for all conditions
Presence of gene may not result in condition
Confirmed presence gives stress/fear
Problem telling/testing rest of family
Discrimination by employers/insurers
Ethics of termination
Could increase intolerance/discrimination of disabled
Define DNA sequencing
The process of working out the order of bases on a DNA molecule/gene
Steps of original Sanger method (chain termination method)
Single stranded DNA, DNA polymerase and DNA nucleotides are mixed with one type of radioactively labelled nucleotides (once added to a sequence, terminate DNA synthesis)
DNA sequences of every possible length were created (each terminating with a radioactively labelled nucleotide)
These were run via gel electrophoresis to work out the sequence on the gene
Disadvantages of original Sanger method
Very slow
Labour intensive
Only suitable for very short genes
Steps of updated Sanger method
Fluorescently labelled nucleotides replaced radioactive ones
All 4 types of labelled nucleotides placed together into a sequencing machine (rather than separately)
Machine runs different lengths of DNA through a gel in a capillary tube (instead of gel electrophoresis)
A laser scans each length and reads the fluorescent base sequence as a sequence of colours (each colour specific to each base) to reveal the sequence
Advantages of updated Sanger method
Much faster
Less labour intensive
Steps of pyrosequencing
Nucleotides washed over the DNA in a specific order
When a complementary nucleotide is present it joins the chain
The addition of a nucleotide to the chain releases energy
The energy activates the protein luciferin
Light released by luciferin is detected and recorded on a pyrogram
If two identical nucleotides are added together then the intensity of the light emitted is doubled
Points about high throughput sequencing methods
Much faster at sequencing whole genomes than the Sanger method
Cheaper
More errors tend to be made
Aims of the Human Genome Project
Work out the order/sequence of all the 3 billion base pairs in the human genome
Identify all the genes
