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
-Without ATP the myosin heads cannot be released from the actin filaments, locking the muscles in a contracted state
-The process is affected by the level of muscle development and the temperature of the surroundings, higher temperatures will speed up the rate of rigor mortis
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
Explain why modification of pre-mRNA enables one gene to give rise to more than one protein (2)
-The exons can be spliced together in a different order
-Therefore producing different sequences of amino acids
Explain how a gene could give rise to more than one protein (2)
Post transcriptional modification of pre-mRNA can give rise to a number of different mRNA molecules, which would be translated into different proteins
Describe the role of decomposers, such as microorganisms, in the carbon cycle (2)
-Decomposers break down organic material from the dead body
-Decomposers respire
-Releasing carbon dioxide into the atmosphere
Describe a procedure for Polymerase chain reaction (6)
-DNA is cut into fragments using restriction endonucleus enzymes
-Add florescent dye to each section of DNA
-Use a micro-pipette to insert the DNA fragments into wells in the agarose gel
-Apply a current through the gel which makes the DNA travel towards the anode
-DNA restriction enzymes travel at different speeds so bands begin to form
Explain DNA splicing
-The non-coding intron sections are removed
-The coding exon sections are joined together
-The resulting mRNA molecule contains only the coding sequences of the gene
Explain alternative splicing
-The exons of genes can be spliced in many different ways to produce different mature mRNA molecules through alternative splicing
-This means that a single eukaryotic gene can code for more than one polypeptide chain
Suggest DNA profiles are compared (3)
-Comparing total number of bands
-Comparing position of bands
-Comparing band size/width
Explain how DNA bands form in gel electrophoresis
-The molecules are separated according to their size / mass
-Different sized molecules move through the gel at different rates
-The tiny pores in the gel allow smaller molecules to move quickly, whereas larger molecules move more slowly
Describe the changes that occur inside a body in the first week after death (3)
-Body temperature falls
-Stiffening of the muscles
-Break down of cells by enzymes in the body
-Putrefaction/bloating
Changes in the RNA of influenza produce new strains of the virus with an altered spike protein
Devise a procedure to determine the similarity of the strains of influenza in saliva samples collected from different people (5)
-Isolate / extract RNA from saliva samples
-Amplify the RNA/DNA by PCR
-Cut the RNA/DNA/ into fragments using restriction enzymes
-Separate fragments using gel electrophoresis
-More similar the {banding / profile} (of the fragments) the more closely related the strain
Explain the effect of ambient temperature on the rate of decomposition (3)
-An increase in temperature will increase the rate of decomposition / decay
-An increase in temperature increases enzyme activity
-An increase in temperature increases growth rate of bacteria /fungi / decomposers
Describe the changes that occur inside a body in the first week after death (3)
-Body temperature falls
-Rigor mortis / stiffening of the muscles
-Autolysis / break down of cells by enzymes in the body
-Putrefaction / discolouration / bloating (1)
Body farms use the bodies of pigs to study the changes in insect species on a body after death
Describe how this study could be carried out (3)
-Standardisation of pigs studied
-Named {environmental condition / abiotic factor} controlled
-Record the presence of different species (of insects)
-At regular intervals of time
A pathologist can use a number of methods to estimate the time of death of a body found at a crime scene.
(a) The pathologist will measure the body temperature and the temperature of the surroundings.
Explain why it is necessary to take these two measurements as soon as possible (3)
-Calculations of time of death are based on {average body temperature
37˚
-Body temperature at time of death will depend on time of day
-Therefore the calculated value for time of death may not be accurate
Describe an investigation that could be carried out to study the effect of temperature on the time taken for the first instar maggot to become a pupa (5)
-Using {a range / at least five} temperatures
-Temperature control e.g. water bath, incubator
-Timing starts when eggs hatch into first instar maggots
-And ends when the (third instar) maggots begin to pupate /
-Several {eggs / maggots} should be used at each temperature
-Providing food for maggots
-Appropriate controlled variable e.g. humidity. mass of food, species ;
-Plotting data on a graph of temp against time (for first instar to become a pupa);
Describe the role of bacteria in process of decomposition (2)
-Bacteria) producing enzymes for digestion / hydrolytic enzyme} ;
-Respiration produces carbon dioxide
The genetic relationship between these two species of grey tree frog has been
studied using DNA profiling (DNA fingerprinting).
A small sample of DNA was taken from each species of grey tree frog. This DNA
was amplified, fragmented and used to produce a DNA profile (DNA fingerprint)
for each species.
Describe how a DNA profile was produced from this small sample of DNA (6)
-Multiple copies of DNA made
-Using PCR / polymerase chain reaction
-Restriction enzymes used to produce DNA fragments
-Gel electrophoresis
-Load the DNA onto the agarose gel
-Use an {electric current / charge} is applied
-Use of dye / fluorescent tag / UV light
Suggest what effect an increase in temperature would have on the rate of
decomposition of these leaves. (4)
-An increase in temperature would increase the rate of decomposition up to an optimum temperature
-Enzymes used in decomposition
-Increased {heat / kinetic} energy results increase in {number of collisions / energy of collisions (between enzymes and substrate) / enzyme-substrate complexes}
-Increased temperature increases rate at which bacteria increase
-Above a certain temperature rate of decomposition would decrease / stop
-Higher temperatures enzymes become denatured OR bacteria killed
