PCR Genotyping Flashcards
(14 cards)
What is found in a PCR reaction tube?
- DNA template containing the original sequence of DNA to be amplified
- 2 single stranded primers which target specifically the fragment to amplify
- Taq DNA polymerase
- Free nucleotides called dNTPs = deoxynucleotide triphosphates of adenine, guanine, thymine and cytosine
- Reaction buffer
What are the 3 steps of the PCR cycle?
- Denaturation at 90-95 degree for approximately 30 seconds —> double stranded DNA melts open into single stranded DNA molecules
- Annealing at 50-60 degree for approximately 30 seconds —> allows primers to find their complementary sequences on the single stranded template and anneal through base complementarity
- Extension at 72 degrees (some Taq use 68) for approximately 60 seconds —> primers synthesise a new strand by providing a short sequence of double-stranded DNA for the Taq polymerase to extend from and build a new complementary strand
Important features of the PCR cycle
- Template strands also tend to reaneal during the annealing phase but the primers are added in excess to out-compete the other annealing events
- 72 degrees is the ideal working temperature for Taq polymerase which adds one by one the dNTPs complementary to the template in the 5’ to 3’ direction
- Taq will stop adding dNTPs when there is no template left to read or when extension time is over. Extension time is determined by the Taq used and the length of the fragment to be amplified (typical time is 1min/kb)
- DNA fragment to be amplified is determined by where the primers bind. Primers anneal to the separate template strands in opposite directions and serve as starting points for replication by the Taq
- By the end of the extension phase, each single strand from the template has been replicated (partially or fully) into double stranded DNA which then serves as a template in the next cycle
What is meant by genotyping?
Determining the genotype of an individual
Used for disease diagnosis, sex determination of embryos, paternity testing, forensics and studies of molecular evolution and population genetics
What are polymorphic sites?
Sites in a genome where the DNA sequence is known to be variable amongst individuals
Types of polymorphism
- SNPs = single nucleotide polymorphism where point mutations arise by errors in DNA replication
- CNVs = copy number variants which are regions of the genome that vary in copy number either due to duplication or deletion. These are classified into STRs or VNTRs
- STRs = short tandem repeats which are 2-7bps that repeat up to 100 times, common examples being GT or CAG repeats
- VNTRs = variable number tandem repeats which are 15-200bps that are repeated up to 100 times
- Transposable elements (TEs) = also known as jumping genes and they insert themselves into chromosomes
What are TEs or jumping genes?
Semi-parasitic DNA sequences of viral origin that can replicate and spread through the host genome
The Alu element in these have been involved in modifying/creating new genes through alternative splicing mechanisms
Background on Alu elements
About 300 base pairs in length and contains an Alul restriction enzyme site that when activated, it makes a copy of itself and this copy is inserted randomly into one of the 46 chromosomes
They don’t insert symmetrically on both homologous chromosomes, their insertion creates a dimorphic polymorphism at a given locus, the transposon is present whilst on the homologous chromosome it is absent
Consequences of Alu elements
Exactly where in a chromosome a transposable element inserts can have great consequences
If inserted in a gene, it can disrupt the coding sequence or the regulatory sequences of that gene. This can lead to production of the wrong protein but in the wrong cell, at the wrong time or in the wrong quantity.
Other Alu insertions in introns of genes for tissue plasminogen activator (TPA) and angiotensin converting enzyme (ACE) are associated with heart disease
What is the PV93-Alu insert?
An Alu insert within the PV92 locus on chromosome 16
The insert is found within an intron and has no effect on the production of encoded proteins- it bears no reference to disease or relatedness among individuals
What are the different forms of PV92-Alu?
The PV92-Alu genetic system is dimorphic = it has 2 alleles inherited and either the Alu element is present at the PV92 locus (+ allele) or absent (- allele)
3 possible combinations exist = +/+, +/- or -/-
The presence or absence of the insert can be easily detected by PCR using primers that flank the PV92 locus, followed by agarose gel electrophoresis to detect the PCR products (called amplicons)
How is genomic DNA extracted?
Take sample of saliva and add 1ml of 8% NaCl then mix
Centrifuge at 10,000 rpm to pellet cells at the bottom of the tube then remove supernatant
What is the process of PCR using the genomic sample?
- After centrifuging and removing supernatant, add Chelex solution to the cell pellet
- Chelex resin beads are negatively charged and are added to trap (chelate) polyvalent metal ions such as Mg2+. It’s important to remove these cations as some are used as co-factors by enzymes that degrade DNA and some inhibit the PCR reaction
- Vortex until the pellet has dissolved then lyse the cells by incubating the sample at 95 degrees for 10 minutes
- The heat lyses the cells so that the DNA is now released and inactivates undesirable enzymes such as DNAses
- Cool the tube on ice for 1 minute then centrifuge again for 10 seconds
- Take some of the supernatant and add PCR mix to it then mix
- This then undergoes thermal cycle programming (the PCR cycle)
Steps to make agarose gel
- Mix agarose and buffer (consist of 1X TAE)
- Microwave to boil until solution is clear (ensure no bubbles form)
- Add safe view when cooled then pour (safe view allows us to visualise the DNA bands)
- Allow gel to set
