Topic 6 Flashcards
PCR Test about
- PCR process involves three key stages per cycle
- In each cycle the DNA is doubled so in a standard run of 20 cycles a million DNA molecules are produced
- Undertaken in a PCR thermal cycler which provides the optimal temperature and controls the length of time spent at each stage
What does a buffer solution do in PCR
Provides the optimum pH for the reactions to occur in
PCR Stage 1: Denaturing
The double-stranded DNA is heated to 95°C which breaks the hydrogen bonds that bond the two DNA strands together
PCR Stage 2: Annealing
The temperature is decreased to between 50 – 60°C so that primers (forward and reverse ones) can anneal to the ends of the single strands of DNA
PCR Stage 3: Elongation
- The temperature is increased to 72°C for at least a minute –> optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules
What goes into the PCR reaction tube?
Buffer solution DNA sample Free nucleotides primers Taq polymerase
Gel electrophoresis is used to…
- separate the DNA according to their length
Gel electrophoresis process
- DNA is paced into a well in a slab of gel and covered in a buffer solution that conducts electricity
- a potential difference is applied across the gel using an electrical current
DNA fragments are negatively charged so move towards the positive electrode - shorter fragments move faster so the fragments separate according to length
Gel electrophoresis: the fragments are visualised
- southern blotting is carried: the fragments are moved to a more stable medium and denatured to expose their base sequences
- The DNA is incubated with a DNA probe complementary to the target DNA sequence needing to be located
- after allowing the probe time to bind to any complementary sequences any unbound probe is washed away
- probes can either be radioactive and viewed in an x-ray or fluorescent and viewed under a UV light
DNA profiling in forensic science
- DNA samples can be collected from crime scenes e.g. hair, saliva, blood, semen, skin cells etc.
- the DNA is isolated from the samples
- amplified through PCR
- these products are then run through electrophoresis
- DNA profiles are then compared to see if any match
if the samples match it links the person to the crime scene
other uses of DNA profiling
- the more bands on two DNA profiles that match, the more closely related –> paternity tests are used to determine the biological father of a child
- DNA profiles can be used on animals and plants to prevent inbreeding which can cause health, productivity and reproductive problems
- inbreeding reduces the gene pool which can increase risks of genetic disorders
- DNA profiling can be used to determine how closely related two individuals are
- the least related individuals are bred together
Virus
- nucleic acids surrounded by protein
- tiny
- no plasma membrane, no cytoplasm and no ribosomes
- DO have nucleic acids
The protein coat around the virus core is called the…
…capsid
virus envelope
- extra outer layer, stolen from the cell membrane of a previous host cell
Attachment proteins
- stick out from the edge of the capsid or envelope
- allow the virus to cling on to a suitable host cell
some viruses carry ____ inside their capsid
- proteins
- e.g. HIV carries an enzyme called reverse transcriptase
nucleic acid core
- either DNA or RNA
- HIV: RNA
How HIV gets into the host
- HIV is spread through infected bodily fluids
- infection of a new host occurs when these fluids come into contact with mucosal surfaces or damged tissue, or are injected into the bloodstream
- one of the most common infection method is through sexual intercourse
How HIV replicates
- viruses can only reproduce inside the cells of the organism it has infected
- HIV replicates inside the T-helper cells and uses the enzymes and ribosomes of the host to replicate:
1) the attachment protein attaches to the receptor molecule on the membrane of the host T-helper cell
2) the capsid is released into the cell where is uncoats and releases the genetic material (RNA) into the cell’s cytoplasm
3) inside the cell, reverse transcriptase is used to make a complementary strand of DNA from the viral RNA template
4) from this, double stranded DNA is made and inserted into the human DNA
5) host cell enzymes are used to make viral proteins from the viral DNA found within the human DNA
6) These viral proteins are assembled into new viruses, which bud from the cell and go on to infect other cells
The latency period
- a time in which the infected individual may experience no HIV symptoms at all
- the HIV has already rapidly replicated during the initial infection period and replication has now dropped
- can last for years
What is AIDS
- HIV eventually leads to acquired immune deficiency syndrome –> without treatment this takes around 10 years
- the immune system deteriorates and eventually fails
- people with AIDS develop diseases and infections that wouldn’t cause serious problems for people with a healthy immune system –> opportunistic infections
when are people with HIV classed as having AIDS
- people with HIV are classed as having AIDS when symptoms of a failing immune system appear or their T helper cells drop below a certain level
opportunistic infections
- people with AIDS develop diseases and infections that wouldn’t cause serious problems for people with a healthy immune system –> opportunistic infections
Sequence of AIDS symptoms
1) lower than normal t helper cell count
- minor infections of mucous membranes (e.g. in nose, ears and genitals) and recurring respiratory infections
2) T helper cell count decreases further:
- patients are susceptible to more serious infections including chronic diarrhoea, severe bacterial infections and tuberculosis
3) very low t helper cell count:
- serious infections, e.g. parasite infections in the brain it is these serious infections that kill AIDS patients, not HIV itself
what bacterium causes tuberculosis?
Mycobacterium tuberculosis
How does TB infection occur?
- when tiny droplets containing the bacteria (e.g. from an infected person’s cough/sneeze) are inhaled into the lungs
- the bacteria are then taken up by phagocytes where they survive and replicate
- most people don’t develop TB straight away - their immune system seals off the infected phagocytes in the tubercles of the lungs
- in the tubercles the bacteria becomes dormant and the infected person shows no symptoms
- later, the dormant bacteria may become reactivated and overcome the immune system, causing TB
- time between infection and development varies - can be weeks to years
AIDS and TB
- the reactivation of TB is more likely in people with weakened immune systems e.g. people with AIDS
TB sequence of symptoms
initial symptoms: fever, general weakness and severe coughing caused by inflammation of the lungs
progressed symptoms: if left untreated damage to the lungs can cause respiratory failure which can lead to death
Can also spread from the lungs to other parts of the body e.g. brain and kidneys, if left untreated can lead to organ failure
pathogen entry routes
1) the skin e.g. through cuts
2) the digestive system e.g. through contaminated food or drink
3) respiratory system through inhalation
4) mucosal surfaces e.g. the inside of the nose, mouth or genitals
Natural barriers that prevent infection
- stomach acid
- skin
- gut and skin flora
- lysozyme
Natural barrier: Stomach acid
- most pathogens from food or drink are killed by the acidic conditions of the stomach
- some survive and pass into the intestines and invade cell in the gut wall causing disease
Natural barrier: Skin
- physical barrier to pathogens
- if skin is damaged pathogens on the surface can enter the blood stream
- the blood clots at the area of damage to prevent pathogens from entering but some may get in before the clot forms
Natural barrier: Gut and skin flora
- the intestines and skin are naturally covered in billions of harmless microorganisms called flora
- they compete with pathogens for nutrients and space
limits no. of pathogens living in the gut and skin - makes it harder for them to infect the body
Natural barrier: Lysozyme
- Mucosal surfaces (e.g. eyes, mouth and nose) produce secretions (e.g. tears, saliva and mucous)
- secretions contain enzyme ‘lysozyme’
- which kills bacteria by damaging cell walls –> causing them to lyse
what is an immune response
- when a pathogen invade the body the antigens on its cell surface are recognised as foreign which activates the cells in the immune system
