Chapter 5 Flashcards
Deoxynucleotide
Made up of organic base, a ribose sugar and phosphate
dNTPs
The building blocks for amplication
DNA Polymerase
an enzyme that catalyzes the synthesis of DNA molecules from nucleoside triphosphates
Taq DNA polymerase
Thermophilus aquaticus
has strong processivity, but lacks proofreading capability, thermal stable
Polymerase Chain Reaction
The repeated serial reaction involving the use of oligonucleotide primers and DNA polymerase that is used to amplify a particular DNA sequence of interest
Denaturation
The alteration of a protein shape through some form of external stress
Annealing
recombin(DNA) in the double-stranded form following separation by heat
Elongation
the synthesis of new strands by making a complete copy of the templates
Extension
achieved by using the loosened nucleotides of each base to grow the complementary DNA strand. The end result is two double stranded products of DNA
Oligo Primer
initiates DNA amplification in a PCR
Agarose Gel
A polysaccharide matrix that functions as a sort of sieve
Gel electrophoresis
a technique used to separate DNA fragments according to their size
Restriction Enzyme
An enzyme (a protein-endonuclease) that has the property to cut DNA at or near a specific recognition nucleotide sequence (restriction site)
Blunt End
A straight cut of restriction enzymes generates blunt ends, where both strands terminate in a base pair
Sticky end
when one end of DNA is longer than the other (typically 4nt) after restriction enzymes
Restriction fragment length polymorphism
Detects the variation in length by a particular piece of DNA
Used to detect a single nucleotide substitution that leads to the disease
Basic components in a PCR
- Taq DNA polymerase
- dNTPs: the building blocks for amplification
- MgCl2 concentration: Mg++ is the cofactor of the Taq polymerase
- A pair of oligonucleotide primers: to initiate the DNA amplification
- DNA template (target DNA): the DNA sequence to be amplified
Thermocycler
- Heating element in a thermocycler is located in the top cover of the sample compartment. It is to prevent condensation of the reaction mixture inside of the cap of the reaction tube.
- Programmable
- Newer generation of the thermocycler is quite fast in heating and cooling cycles
- Easy to operate
Restriction Enzymes and their function
Enzymes used to cut DNA. They bind to specific DNA sequences and then cleave the DNA at two defined locations, on on each strand
Amplicons
amplified DNA fragments, called amplicons, are separated on an agarose gel using gel electrophoresis
Friedreich Ataxia (FRDA)
- Friedreich ataxia is a neurodegenerative movement disorder
- Autosomal recessive transmission
- The disease is caused by a triplet expansion; GAA trinucleotide expansion within the 1st intron of the FXN gene
- GAA expansion can be detected easily using PCR followed by gel electrophoresis
- Onset of the disease is between 10 and 15 years
- Initial symptoms may include unsteady posture, frequent falling, and progressive difficulty in walking due to impaired ability to coordinate voluntary movements (ataxia)
- Affected individuals often develop slurred speech, characteristic foot deformities, and an irregular curvature of the spine (scoliosis)
Single Nucleotide Polymorphisms
(SNP, pronounced snip) is a DNA sequence variation occurring when a single nucleotide adenine (A), thymine (T), cytosine (C), or guanine (G]) in the genome (or other shared sequence) differs between members of a species or paired chromosomes in an individual
Hereditary Hemochromatosis (HH)
- Hereditary hemochromatosis is an autosomal recessive disease
- The disease makes patient’s body to absorb too much iron (Fe) from the food you eat. The large amount of iron is stored in patient’s liver, heart and pancreas. Excess amount of iron can poison your organ and can lead to cancer, heart disease and cirrhosis.
- It is caused by mutation in the gene called HFE
- One type of the mutations of HFE gene is a substitution of one amino acid at position 282 – cysteine to tyrosine (UGS to UAC; DNA code is TGC to TAC)
- The mutation creates a ne RsaI site (5’ GT↓AC 3’). Therefore, it can be detected by PCR, followed by a restriction enzyme digestion and gel electrophoresis
