PCR and electrophoresis Flashcards
what is PCR and what can it be used for
-polymerase chain reaction used to make multiple copies of DNA fragments
-amplified DNA can be then analysed e.g used in forensic DNA analysis (genetic profiling) or analyses including genetic diseases
what are the similarities between PCR and DNA replication
differences
-only short sequences can be replicated not entire chromosome in PCR
-requires addition of primer to make process start
-a cycle of heating and cooling is needed to separate DNA, bind primers and replicate DNA
similarities
-both need supply of free nucleotides
-both use enzymes
what are the raw materials required for PCR and why
-sample of DNA to be copied
-supply of each type of DNA nucleotide containing bases A,C,G,T
-short pieces of single stranded DNA= primers= act as signals to DNA polymerase to start copying
-magnesium ions= cofactor for Taq enzyme
-Taq DNA polymerase
-PCR machine/ thermal cycler
Why is a Taq DNA polymerase enzyme used instead of DNA polymerase
-thermostable/ do not denature at 95 degrees
-so PCR can be cycled repeatedly without stopping to reload enzyme
explain the steps of PCR
STRAND SEPARATION= 95 degrees
1.DNA sample is mixed with DNA nucleotides, primers, magnesium ions and Taq DNA polymerase
2. mixture is heated to 95 degrees= breaks hydrogen bonds between comp base pairs, causing DNA to denature the double strand into single stranded DNA
PRIMER BINDING= 55 degrees
3. mixture cooled to 55 degrees so primers can anneal (bind by hydrogen bonding) to one end of single DNA strand= gives small section of double stranded DNA at ends of each single strand
4. Taq polymerase can now bind to end where there is a double strand
STRAND SYNTHESIS: 72 degrees
5. temp raised to 72 which keeps DNA as single strands
6. Taq catalyses addition of DNA nucleotides to single stranded DNA= starts at end with primer and proceeds in the 5 prime to 3 prime direction
7. when Taq reaches the other end of DNA molecule, new double stranded DNA is generated
=whole process begins again and is repeated for many cycles
what are examples of application of PCR
-Tissue typing – reduce risk of rejection of the transplant
-Detection of oncogenes (gene with potential to cause cancer) – allows medication to be tailored to the patient
-Detecting mutations – screening of parents for recessive alleles, prenatal screening of foetal cells from mother’s blood stream, or during IVF
-Identifying viral infections – detect small quantities of viral genome e.g. HIV or Hep C
-Monitoring the spread of infectious disease
-Forensic science – small quantities of DNA amplified for -DNA profiling to identify criminals
-Research – e.g. amplifying DNA from extinct or ancient sources such as Neanderthal or woolly mammoth. Analysis of cells or tissues to find out which genes are switched on or off
what is electrophoresis
Separating DNA
-Different sized fragments of DNA
-The fragments can differ by only base pair
-For identification and analysis
what are the principles of electrophoresis and how does it work
-Uses an agarose gel covered by a buffer solution
-Electrodes placed either end of the tank so an electric current can pass through the gel
-DNA is negatively charged (due to the many phosphate groups)
-DNA fragments migrate towards the positive electrode (anode)
-Smaller fragments travel faster, so will travel further in a fixed time
explain the steps of electrophoresis to separate DNA fragments by size
1.Samples of DNA are cut into fragments using restriction enzymes
2.DNA samples are then loaded into wells cut into an agarose gel
3.The gel is placed in a tank containing a buffer solution, electrodes are attached and a voltage applied
4.DNA is negatively charged as a result of phosphate groups
5.DNA therefore travels towards the positive electrode (anode)
6.The movement of DNA fragments is inversely proportional to molecular size (smallest travel furthest)
how can proteins be separated and for what
Same principle as for separating DNA fragments
Uses a charged detergent such as sodium dodecyl sulfate (SDS), equalises surface charge on the protein so they can separate according to size (molecular mass) as they move through the gel
Can be used to analyse types of haemoglobin proteins for diagnosis of conditions such as sickle cell anaemia, aplastic anaemia or leukaemia
what is used to make DNA fragments visible
-radioactive labels
-uv light
-visible stain
