Topic 6 Humphries Flashcards
What happens when no documents are found on a dead body?
forensic techniques are used: fingerprinting, dental records, DNA profiling
How can you get fingerprints?
using aluminium, carbon or iron powders
using superglue (reacts with water)
using ninhydrin (reacts with amino acids)
magnets and iron flakes
once obtained are examined and compared - need 16 identical points for a match
When are dental records used?
when there’s no fingerprints on file or the body is damaged
Why can we use dental records?
teeth decay very slowly and are more resistant to burning. Can make teeth chart and compare with missing persons or look at growth and get an age range
Issues with fingerprinting and dental records
fingerprints may be partial or not present
dental records need to be up to date and if no records then only age range is gathered
Exons
coding regions of DNA
Introns
non coding regions of DNA
short tandem repeats
short DNA sequences within introns that are repeated many times
Every person’s DNA is…
unique (except twins)
Why is everyone’s DNA being unique useful?
because patterns in the nucleotide sequence can be used to identify individuals - in DNA profiling STRs in non coding regions of DNA are used
What do introns allow?
crossing over to occur and therefore variation in a population (this is essential for species survival) - they do this by creating space between exons
A gene is made up of…
introns and exons but only the exons are expressed
How long can repeat sequences be?
between 2 and 50 base pairs
How many times can an STR be repeated?
from 5 to several hundred repeats
The same STRs occur at the same…
locus on a pair of homologous chromosomes but there may be different numbers of repeats
What are the stages of DNA profiling?
- obtain sample
- extract DNA
- cut up DNA (to collect specific STRs)
- copy DNA (PCR)
- separate DNA (gel electrophoresis)
- visualise DNA (southern blotting)
Obtaining sample of DNA
- collect cells
- physically break down sample in a buffer solution, salts and detergent, this disrupts the cell membrane
- separate the DNA from the rest of the cell debris by filtration and centrifugation
- add protease enzymes to remove proteins and cold ethanol to precipitate out the DNA
- wash the DNA sample in buffer solution
Restriction enzymes are also known as…
restriction endonucleases
Blunt ends
in line
sticky ends
jagged, normally staggered ends
What is a restriction endonuclease?
an enzyme that cuts DNA at specific sites - cuts phosphodiester bonds in polynucleotide chain
Where are restriction sites?
this is where the enzyme cuts the DNA, they’re either side of an STR so the repeated sequence remains intact but the fragment is separate from the rest of the DNA
What is the polymerase chain reaction (PCR) used for?
is a technique for the amplification of DNA (copying many times)
What do you need for PCR?
- DNA
- DNA nucleotides
- primer
- Taq polymerase
How does DNA grow in PCR?
the number of copies of DNA grows exponentially
Why is PCR useful?
is only a small amount of DNA found at the crime scene then a greater quantity can be made for tests and experiments
Buffer
suitable chemical environment - optimum pH
Template
DNA with sequence to be amplified
DNA mononucleotides
to make new DNA
Primers
short DNA sequences that specify sequence to be amplified (complementary to sequence we want)
Polymerase
enzyme that catalyses synthesis of DNA - Taq polymerase is used as it can work at high temperatures
Why is DNA heated in PCR?
to break the hydrogen bonds
What’s the purpose of cooling the DNA sample?
primers can bind to complementary sequence
What characteristics of polymerase prevent it from being denatured?
strong bonds
hydrophilic/phobic interactions
What are the 2 stages of PCR and the temperatures?
- denaturation, 95°C
- annealing, 55°C
- elongation, 70°C
Stages of gel electrophoresis
- DNA placed in wells of an agarose gel (stable medium for fragments to move)
- Gel is submerged in a buffer solution and connected to electrodes that produce a p.d across gel
- negatively charged fragments of DNA migrate through the gel according to charge and size
- smaller fragments move further in a given time so they end up closer to positive electrode
- a reference sample with fragments of a known length may be added for comparison
method of visualisation
southern blotting
How does southern blotting work?
- DNA is transferred to a membrane (nylon or nitrocellulose) from the gel
- the membrane is incubated with a specific DNA probe (a short section of DNA that has a complementary base sequence to DNA of interest)
- the DNA probe will be labelled with either radioactive or fluorescent markers to the bands of interest can be visualised
How are radioactive markers seen?
using x-ray film
How are fluorescent markers seen?
UV light
DNA ladder/marker
digested DNA with known base pair length
Uses of DNA profiling
- medical diagnostic tests (see if you have a disease)
- forensics (crime investigations)
- paternity testing
- analysis of ancient DNA (evolutionary relationships)
- cloning genes
Why is DNA profiling so useful?
- if no eyewitnesses, then gives good evidence
- DNA is unique to each person (except twins)
When DNA profiling becomes a problem?
- if individuals being tested are closely related
- many stages so lots of contamination could occur
- only a few repeated sequences are tested so less likely to be unique
microorganisms
bacteria and fungi are responsible for breaking down and digesting decaying matter
How do microorganisms break down decaying matter?
They release enzymes into their surroundings which digests the organic matter so that they can then absorb those organic molecules to use in respiration. The carbon is converted into CO2 and released into the atmosphere
Organic matter
carbohydrates, proteins, lipids, nucleic acids = great energy source for decomposers/ microorganisms –> multiple make more decomposers
Codon
sequence of 3 nucleotide bases on an mRNA molecule that codes for a particular amino acid
degenerate
each amino acid is coded for by more than one triplet
non-overlapping
no base of one triplet contributes to part of the next triplet
DNA sense strand
coding strand
DNA antisense strand
template strand
anticodon
a sequence of 3 nucleotide bases on a tRNA molecule which is complementary to the corresponding mRNA codon
mRNA splicing
Between transcription and translation mRNA is edited - some sections are removed, others spliced together. Non-coding introns are removed meaning exons can join together differently which means several proteins can be formed from one length of mRNA
pre-mRNA
allows mRNA splicing to occur so that some parts can be removed and the different ways exons bind together means more than one protein can be made
spliceosome
introns are removed by this - enzyme that does splicing
Antibiotics
- are substances that can inhibit growth or destroy bacteria
- produced by other living organisms
- effective against prokaryotic cells but leave eukaryotic cells alone
- cannot be used to kill or inhibit growth in any other microbes including viruses and fungi
What are the two types of antibiotic
bactericidal and bacteriostatic
Bactericidal
directly destroys bacterial cells
Bacteriostatic
inhibit growth of bacterial cells (don’t kill ones already there only stop replication)
Disruption of the cell membrane - changes in permeability that leads to lysis
bactericidal
Inhibition of cell wall synthesis - weak wall can lead to lysis
bactericidal
Inhibition of specific enzymes found in the bacterial cell but not the host
bacteriostatic (unless enzymes are essential)
Inhibition of protein synthesis meaning enzymes and other proteins not produced
bactericidal
Inhibition of nucleic acid synthesis, replication and transcription. Prevents cell division and or synthesis of enzymes
could be either
Why is antibiotic resistance common?
- they can replicate quickly so higher chance of mutation occurring
- bacterial population sizes are usually in the billions = not just one cell containing mutation
- random mutations = some will be advantageous to the cell = survival against antibiotics (antibiotics act as selection pressure)
Evolutionary race
are our medicines evolving faster than bacteria (we’re not winning cause we’re worried)
Bacterial mutations
- Mutations are random and rare
- Mutations are passed on vertically but also by horizontal gene transfer
Horizontal gene transfer
conjugation between bacterial species means antibiotic resistance is not contained to one sole species
bacterial cells can now also be resistant to a range of different antibiotics
Tuberculosis
is an infectious disease that can affect any part of the body but is usually found in the lungs - Mycobacterium tuberculosis
Symptoms
- fever
- night time sweating
- loss of weight
- persistent cough
- constant tiredness
- loss of appetite
- coughing up blood
How is TB caught?
it is caught by aerosol infection (inhaling). The bacteria lodge in the lungs and start to multiply - they destroy lung tissue and create cavities
To diagnose TB:
- chest x-rays
- sample of sputum coughed up is taken and cultured to see what bacteria manifests
- blood tests (T cells with antigens specific to Mycobacterium tuberculosis)
- skin test (small amount of tuberculin injected, if inflamed then TB is present)
Primary infection
- can last several months with no symptoms
- person infected deals with the infection by their macrophages engulfing the bacteria
- from this masses of tissues forms which have dead bacteria and macrophages within it
- these tissues are tubercules
- after 3-8 weeks the infection is controlled and the lung heals
Active TB
- occurs when previous infection is no longer controlled or if the body is overwhelmed by a large amount of bacteria
- bacteria multiply rapidly, breaking down tissue in the lungs causing spaces where alveoli should be
- the bacteria target T cells and therefore reduce the production of antibodies
- eventually TB causes death
When will TB reactivate?
when the immune system is weakened and cannot contain TB. Normally due to old age or the very young or malnutrition and poor living conditions
Effect of TB
slows down gas exchange due to a smaller surface area
How does TB evade the immune system?
- the TB bacteria have a thick waxy outer layer which protects them from the macrophage enzymes. They will lie dormant in the tubercules until the sufferer has a weakened immune system
- TB can disable 2 of the mechanisms a macrophage uses against bacteria: disrupts production of cytokines, stops the apoptosis of the macrophage
Granuloma
area of inflammation
We’re in an evolutionary race with pathogens
the human immune system is the main selection pressure so they’re evolving to create ways to evade the immune system
Antibiotic presence is a selection pressure
- bacteria without resistance gene, selected against and destroyed
- bacteria with resistance gene, survive, grow, divide
Multiple resistance
some bacteria have developed resistance to several antibiotics which causes problems in treatment as new drugs or new combinations need to be found
Hospitals are now taking procedures to tighten up infection control and antibiotic prescribing procedures:
- wash hands
- signs to remind you
- no ties, long sleeves, watches, etc
- Only when diagnosed as a bacterial infection do antibiotics get prescribed and the course must be completed
