CMB2000 Essential Biomedical Research Skills Flashcards
What is PCR
Polymerase Chain Reaction
What does DNA replication require?
-Template DNA
-40 or more proteins
-Helicase
-Primase
-Polymerases
-Nuclease
-Ligase
-ssDNA binding proteins
-Sliding clamps
What are the advantages of PCR?
-Sensitive (can amplify as little as one molecule)
-Specific (cam amplify a unique target sequence)
-Cheap
-Rapid
-Robust (DNA is very stable)
What’s required for PCR?
-Template dsDNA
-2 primers (small ssDNA)
-Polymerase (copies the template)
-dNTPs
-Magnesium (cofactor for DNA polymerase enzyme)
-Buffer (maintains pH and provide necessary salt)
What is the function of the sliding clamp in DNA replication?
The clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand.
Describe the primers used in PCR?
-One for template and for complementary strand
-Single stranded DNA
-Length of 18-24 bp
-40-60%G/C content
-Start and end with G/C pairs
-Melting temp of 50-60C
What must primer pairs not have to prevent primer dimers?
Complementary regions to each other
Why is Magnesium required in PCR?
Magnesium acts to enhance the enzymatic activity (specifically of DNA polymerase) thereby supporting DNA application
What makes up the buffer found in PCR?
-pH of 8-9.5
-Tris HCl
-Potassium ions (KCl) promotes annealing
What are the 3 stages of PCR?
-Denaturation
-Annealing
-Elongation
What is Nancy520 an example of?
An Intercalating agent, which aids in detection of DNA.
How may we detect PCR products?
Run products on agarose gel, using intercalating dye to stain DNA to determine size and yield.
Give some key steps involved in cloning
-Bioinformatics searching
-Design primers
-PCR
-Choose plasmid and insert
-Transform into competent E Coli
-Select correct colonies
Give some uses of PCR in biotechnology
-Cloning
-Manipulating DNA
-Knock out genes
-Fuse host proteins
What must our template DNA for PCR be?
-Clean and pure
-Contaminent free
-High concentration
Describe reverse transcriptase PCR
-Convert RNA to cDNA, using reverse transcriptase (a retroviral enzyme) that converts RNA to DNA
-Amplify DNA by PCR (including qPCR)
How does qPCR/Real time PCR work?
-Using a fluorescent report in the PCR reaction
-Couples amplification of the sequence with quantification of the concentration of DNA
How does qPCR using SYBR green work?
Fluorescent that binds to groove of dsDNA
How does qPCR using TAQman work?
Fluorescent that uses probes with fluorescent reporter and quencher
In qPCR, if there is a time to reach the cycle threshold, what does this signify?
more cDNA in the sample
What may we use as reference genes in qPCR?
-House keeping genes
-As these have a constant level of expression, which is not affected by experimental factors
Why do we use reference genes in qPCR?
-Essential to support validity of qPCR
-Confirms RNA extraction was good and efficient
Give some common uses of PCR in diagnosis.
-Genotyping the patient
-Genotyping the pathogen
-Phenotyping the disease
Why may we use PCR to genotype a patient?
-Diagnose genetic traits
-Detection of carriers of genetic traits
-Tissue matching (HLA typing)
-Pharmacogenetics