PCR

Question 1

PCR origins

Answer 1

PCR Origin
allsed to determine har changes in genome sequence lead to dounstream changes in gene expression and physiology
• Kary Mullis - was awarded a Nobel Prize in Chemistry in the year 1993 for inventing the PCR technique
• PCR is a method of DNA cloning
• It is a rapid method that does not require host cells for DNA replication
• Can amplify target DNA sequence from tiny amounts of DNA
-Used to determine how changes in sequence lead to downstream changes in gene expression and phenotype

Question 2

PCR Components

Answer 2

• Template DNA
• Primers - short oligos 20 - 25 nt in length
• Artificially designed
• some aspects of the sequence to be amplified must be known
• dNTPs - deoxynucleoside triphosphates
• free nucleosides that can be incorporated into the replicated DNA fragments by use of the polymerase

-DNA polymerase - an enzyme that is involved in replicating the target DNA fragments
* It must be heat stable so that it can remain functional during thermocycling
-DNA polymerases - isolated and cloned from thermophilic bacteria such as Thermophilus aquaticus (Taq) and Pyrococcus furiosis (Pfu) are used in PCR

-Other components - Mg2* or ions are co-factors or buffer pH regulator

Question 3

Cofactor

Answer 3

It helps the active site of an enzyme be exposed to the substrate or allows it to conform. It is mostly inorganic

Question 4

Oligo

Answer 4

Another word for primer and means the nucleotide is artificially synthesized

Question 5

Amplicord

Answer 5

It is the final product in test tube after PCR occurred

Question 6

PCR process

Answer 6

PCR requires two primers
• Primers: Short, single-stranded oligonucleotides, one complementary to 5’ end and another complementary to 3’ end
• dsDNA to be cloned is put in tube with DNA polymerase, Mg?+, and dNTPs
• Primers anneal to denatured DNA
• Complementary strands are synthesized by heat-stable DNA polymerase
• e.g. Taq DNA Polymerase isolated from thermophilic bacteria

Question 7

PCR steps

Answer 7

• Three steps of PCR
• Denaturation - heat to 94 - 95°C to allow DNA template strands to separate
• Primer annealing (hybridization/annealing) - reduce temperature to Tm - 5°C to allow for primer binding
• Extension - Temperature suitable to the polymerase (72°C) to allow the strand to be extended
• Steps are repeated over and over using thermocycler to amplity DNA exponentially
• DNA strand is doubled in each cycle
• New strands along with old strand serve as templates in next cycle

Question 8

Target DNA Amplification

Answer 8

• Target DNA sequence doubles in each cycle
• Number of copies of the
fragment = 2”
• Where n = number of cycles
• By 25 cycles, you have 225 = approx. 33,5 million copies in the reaction tube

Question 9

Setting up a thermocylcer

Answer 9

• Thermocycler is set up to have an initial Denaturation step that allows for double stranded DNA to separate.
• It then cycles through denaturation, annealing and extension for between
25 - 35 cycles.
• There is a final elongation step to ensure that all strands are replicated prior to termination.
• The reaction is terminated by reducing the temperature to 4°C.
• PCR products are stored by freezing samples at -20°C.

Question 10

Setting up a thermocylcer

Answer 10

• Thermocycler is set up to have an initial Denaturation step that allows for double stranded DNA to separate.
• It then cycles through denaturation, annealing and extension for between
25 - 35 cycles.
• There is a final elongation step to ensure that all strands are replicated prior to termination.
• The reaction is terminated by reducing the temperature to 4°C.
• PCR products are stored by freezing samples at -20°C.

Question 11

Capillary electrophoresis

Answer 11

• Capillary electrophoresis is a gel free method for nucleic acid separation
• Uses the property of the capillary tube - diameter of approximately 50 um
• Fragments move through the capillary tube at different speeds due to their electrophoretic mobility
• Positively charged particles move fastest and negatively charge particles move slower as their movement is impeded by the anode
Duke to negatively charged phosphate backbone, larger DNA fragments move slower than smaller ones
• A detector placed along the capillary tube can detect UV or fluorescent signatures in the tube
• These measurements are recorded over time and represented as an electropherogram
It is faster than agarose gel, less labor intensive and does not need buffers

Question 12

Negative PCR control

Answer 12

It contains all the same substances that a normal PCR has except for DNA.
It checks for contamination.

Question 13

PCR limitations

Answer 13

• Some information about nucleotide sequence of target DNA is required to synthesize primer
• Minor contamination from other sources can cause problems (e.g., skin cells from researcher)
• PCR cannot amplify long segments of DNA (maximum 10 kb)
- problem with primers: If a target is rich in CG, primers may anneal too strongly and if it is rich in AT then it anneals too weakly

Question 14

PCR Applications

Answer 14

PCR is the most widely used technique in genetics and molecular biology
• Allows for screening of mutations involved in genetic disorders
• Location and nature of mutation can be determined quickly
• Allele-specific probes for genetic testing can be synthesized, making PCR important for diagnosing genetic disorders
• It is used in genotyping and genetic variation studies
• It is also used for gene expression studies

Question 15

Variations to PCR

Answer 15

Variations to Standard PCR
• Reverse Transcriptase PCR (RT-PCR)
• PCR technique modified to amplify RNA rather than DNA
• Involves reverse transcription of RNA to cDNA
• Real Time/quantitative PCR (qPCR)
• A quantitative method that relies on fluorescent probes to determine the amount of DNA/copy number that is present in a sample.
• This is often combined with RT-PCR to quantify mRNA expression
• Multiplexing
• Multiple PCR products are amplified in one reaction tube
• They are individually labelled with probes bound to primers and detected on a gel or via capillary electrophoresis

Question 16

Reverse PCR

Answer 16

During RT-PCR, the starting material is RNA
• The RNA can be reverse transcribed to form
CDNA
• The image on the right shows how mRNA containing polyA tails are converted to cDNA
• MRNA Is first incubated with an oligo(dT) primer that can bind to the polyA tail

• A reverse transcriptase binds to dsRNA and polymerises the cDNA strand
• RNA in the dsRNA/DNA hybrid is then partially digested with RNAse H
• A DNA polymerase is added to synthesise the full dsDNA strand
• Gaps are then sealed with a DNA ligase

Question 17

RT-PCR

Answer 17

• RNA samples can be converted to cDNA using reverse transcription
• For mRNA to cDNA conversion, oligo(dT) primers are useful
• Random hexamers can be used to convert total RNA to cDNA
• These cDNA samples are then used in standard PCR reactions
• Rather than DNA however, the researcher is detecting and quantifying the amount of each RNA fragment that was in the original sample

Question 18

qPCR

Answer 18

Real Time PCR is also known as quantitative PCR (qPCR)
• The amount of DNA present at the end of the amplification cycle is proportional to the amount of target DNA
• If you are using a cDNA sample and want to differentiate between mRNA expression levels, this technique is useful
• Probes that bind to the middle of the target DNA sequence are added to the reaction
• These probes are designed to contain a reporter dye and a quencher
• When the probe is intact, the reporter dye signal is quenched
• A polymerase that extends the peptide chain will cleave the reporter dye, allowing for signal emission
• This signal is then quantified and recorded after each cycle

Question 19

Multiplex PCR

Answer 19

Multiple PCR reactions (can be >10) are carried out in the same reaction tube
• Usetul it you need to do multiple PCR reactions
on the same target
DNA sample, especially useful when source DNA yield is low
• Allows to save on costs of multiple PCR reactions and is fast
• Can be used to detect multiple different genetic loci in one reaction
• Commonly used in forensic DNA profiling to detect numerous STR loci in one experiment
• Primers must be carefully selected to have the same/similar annealing temperatures
• Two different annealing temperatures can be alternated during cycling

Question 20

Multiplex PCR precautions

Answer 20

Genomic loci must be carefully selected in order to ensure that fragments detected during the PCR reaction do not overlap
• After the multiplex PCR reaction, products can be resolved on an agarose gel or by capillary electrophoresis
• Sizes of the fragments should be significantly different so as to not overlap with each other during electrophoresis

Question 21

How to interpret an electropherogram

Answer 21

There are always two alleles at each locus, so you should have a maximum of two peaks
• One peak suggests that the individual is homozygous at the locus as both alleles have the same number of repeats.

Question 22

Forensic DNA profiling

Answer 22

Multiplexing is used in forensics to detect multiple short tandem repeat (STR) loci in one reaction
• This form of forensic analysis is useful for tiny DNA samples or damaged DNA as you are detecting certain regions of the genome rather than performing whole genome sequencing
• However, the limitation is that the fewer markers that are used in the study, the lower the probability that your sample matches the killer
• For example, measure the allele sizes of one STR locus in a DNA sample from Person A. On the two alleles at the locus, one has 10 repeats and the other has 11. Therefore the genotype at the STR locus is 10/11 for person A.
• Now assume that the 10/11 genotype is present in 1/5000 individuals in the population.
This would suggest that there is a high probability that the DNA sample may belong to another individual, including the ones that match on your list of suspects.
• Therefore, it is important that many STR loci are analysed for a DNA analysis in order to reduce the probability of a false positive.

Question 23

CODIS database

Answer 23

• The COmbined DNA Index System (CODIS) was developed by the United States government in order to link biological evidence to crime scenes
• 20 common STR loci were selected and developed into commercial kits for STR analyses by multiplexing
• A minimum of 13 matching STR loci are required to convict a criminal
• In the United Kingdom, 10 STR loci are sufficient
• Multiplexing combined with capillary electrophoresis is used to create electropherograms that are used to genotype the samples
• Primers may be fluorescently labelled with different colours in order to facilitate adequate detection and differentiation by capillary electrophoresis if the allele sizes are similar

Question 24

cDNA

Answer 24

Complemetary DNA
-It has even transcribed from a specific mRNA using enzyme reverse transcriptase

Question 25

qPCR detects initial amount

Answer 25

As amplification occurs, fluorescence is released as quenchers are removed
-At the end each cycle of PCR, the fluorescence signal increases and is measured and the fluorescence detector monitors the fluorescence intensity.
-The cycle at which the fluorescence intensity reaches a set threshold (Ct value) is inversely proportional to the initial amount of target DNA as more initial DNA leads to earlier Ct values due to faster amplification
-Compare Ct value to standard curve with known DNA amounts to determine initial DNA