Topic 6: Forensics, PCR and electrophoresis, Splicing Flashcards
call me a detective n dat
Why do we use PCR?
-Fast and inexpensive way to amplify small segments of DNA
-Used to copy a specific gene or portion of a gene, so that we can study the function of that gene or gene region
-Primers are used to highlight the regions you want
Explain the PCR process (polymerase chain reaction)
-95 degrees denaturation step. First, DNA is heated to a high temperature so that the 2 strands separate
-Annealing step (50-60c). Temperature is reduced so that DNA primers bind to either end of the template. There are 2 primers, one to bind to each strand of DNA
-Extension step. Temperature is raised to 72c to activate Taq polymerase, which elongates the primer for template strand
Explain how we can use temperature to determine time of death
-Mammals produce heat via metabolic reactions e.g. respiration
From TOD, these reactions slow down and stop so body temperature falls until it is the same as the surrounding temperature
-Scientists know rate of cooling of human body is 1.5-2 degrees per hour so we can calculate how long the person has been dead
-Amount of clothes the person is wearing will affect the, weight and air temperature
Explain how we can use degree of muscle contraction to determine TOD
-4-6 hours after death, muscles contract and become stiff
-Muscle cells become deprived of oxygen so they have to respire anaerobically and this causes lactic acid build-up
-This decreases the PH and inhibits enzymes that produce ATP
-No ATP means actin and myosin become fixed so the body stiffens
-Smaller muscles contract first, affected by degree of muscle development and temperature
Explain how we can use entomology to determine TOD
-When someone dies, they are quickly colonised by insects, flies are usually the first to appear, beetles come later
-TOD also estimated by life cycle of of insects e.g. blowfly larvae lay eggs. If there is only eggs in a body, assume it has been less than 24 hours
-Affects insects life cycle: drugs, humidity, oxygen and temperature
Explain how we can use the extent of decomposition to determine TOD
Hours - few days = Cells and tissue broken down by the body’s enzymes and bacteria, skin is greenish colour
Few days - few weeks = Microorganisms decompose the body, these organisms produce methane so the body becomes bloated, and skin blisters
A few weeks = Tissues turn liquid and seep out the body - putrefaction
Few months - few years = Only the skeleton is left
Decades centuries = Skeleton begins to disintegrate until there is nothing left
Explain what happens directly after death (succession) after death
-As bacteria decompose the tissue, this makes the conditions more favourable for flies and larvae
-When flies and larvae are on the dead body, this makes it favourable for beetles
-When tissue remains, not favourable for most organisms
-As the body dries out it is less favourable for flies but beetles can decompose dry tissue so they remain
Describe how one PCR cycle would increase the quantity of DNA present (3)
-Heat to 90 degrees to break hydrogen bonds between DNA strands
-Annealing stage at 50-70 degrees
-Extension stage
-To double the quantity of DNA
Explain the role of DNA primers in production of amplified base sequences (2)
-Primers have a specific base sequence
-They bind to complementary bases at either end of the DNA to be amplified
-Providing a site for the DNA polymerase to bind
Devise a procedure, using electrophoresis, to compare amplified DNA (4)
-Use restriction endonuclease enzymes to cut DNA into fragments
-DNA samples are loaded onto gel
-Electric current is passed through the gel
-Dyes are added to visualise the bands
-The position of the bands produced can be compared
The DNA produced by PCR was analysed to find out if a black panther was involved
Explain how gel electrophoresis could be used to find out if this DNA came from a black panther (5)
-Use agarose gel and load it into a tank
-Pass a current through the gel
-Use a dye to stain the DNA
-Use DNA of black panther
-Compare DNA profiles
-A match would indicate that DNA from a black panther was present
The quantity of worm DNA in the beetle gut was very small
Describe how sufficient DNA was produced to carry out the analysis (3)
-Polymerase chain reaction
-The PCR is repeated multiple times
-Denaturation happens at around 90 degrees and annealing happens around 50-70 degrees
Suggest how useful the following additional sources of evidence would be for a pathologist in determining the time of death.
Body temperature (2)
State of decomposition (2)
-When humans die, metabolic reactions which produce heat stop, so body temperature falls. Rate of cooling is 1-2 degrees every hour after death
-Body size, environmental temperature and clothing all affect temperature drop
-As time goes on, body decomposes
-Environmental temperature affects decomposition
Gene expression leads to the synthesis of proteins that give rise to the phenotype
Describe how one gene can give rise to more than one protein (3)
-Introns are removed
-Exons can then be arranged in several different ways (alternative splicing)
-This produces different mRNA from the same gene
-Translation of mRNA gives different amino acid sequences
The RNA produced during transcription is known as pre-mRNA. Pre-mRNA can be modified before being translated on the ribosome
Describe how the pre-mRNA may be modified before being translated on the ribosome (2)
-pre-mRNA contains exons and introns
-introns are removed and exons are spliced together to produce a strand of mRNA
