Restriction Enzymes and PCR-RFLP AnalysiS, Primer Design, and BLAST

Restriction Endonuclease

• An enzyme isolated from bacteria that cuts DNA molecules at specific sequences. They recognize the base pairs within the DNA • Isolation of the enzyme is critical to the development of recombinant DNA technology and genetic engineering

Werner Arber

provided the theoretical framework that described the biology of restriction and isolated the first type I restriction enzyme, EcoBI

Hamilton O. Smith

discovered the first type II restriction enzyme, endonuclease R

Daniel Nathans

demonstrated how powerful restriction enzymes could be when used for the physical mapping of SV40 DNA and its genes

Palindromic

sequence is identical when read in opposite direction in the complementary strand

STICKY ENDS

Staggered ends on a DNA molecule with short, singlestranded overhangs. AKA cohesive ends, have unpaired DNA nucleotides on either 5’- or 3’- strand which are known as overhangs

BLUNT END

A straight cut, down through the DNA that results in a flat pair of bases on the ends of the DNA. AKA non-cohesive ends, since there is no unpaired DNA strand fleeting at the end of DNA

Extensive metabolizers

Genotype: C/C

Alleles: Individuals carry 2 functional alleles

Phenotype: Patients are likely to metabolize drugs normally and medication is likely to work as intended

Ultrarapid metabolizers

Genotype: T/T

Alleles: There are 2 copies of a gene with increased activity

Phenotype: Patients metabolize medicines too quickly to experience relief from symptoms or disease. Certain drugs should be used with caution (e.g., opioids

Intermediate metabolizers

Genotype: C/T

Alleles: Individuals carry 1 defective allele

Phenotype: Patients may have slower drug metabolism which may impact breakdown of medicines causing effects similar to poor metabolizers but not as pronounced

Poor metabolizers

Genotype: T/T

Alleles: There are two defective alleles resulting in substantially decreased drug metabolism and higher levels of drugs

Phenotype: Patients experience a very slow breakdown of medications making side effects more pronounced

Buffer

neutralize DNA polymerase inhibitors present in blood

Deoxyribonucleoside triphosphates:

building blocks of DNA

Magnesium

cofactor for enzyme activity

Polymerase:

responsible for the elongation of new DNA strands

Primers

DNA fragments that recognize and bind to target DNA

Salts

provide the correct ionic strength

Template DNA

double stranded helix containing genetic code

Primer Length: 18 to 30 nucleotides

Long enough: adequate specificity

Short enough: primers to bind easily to the template

Primer Length: __ to __ nucleotides

Long enough: adequate ____________

Short enough: primers to bind easily to the _____________

GC Content: 40% to 60%

Calculated by dividing the sum of G and C by total number of bases present in the primer

GC Content: __% to __%

Calculated by dividing the sum of _ and _ by the total number of _ present in the primer

Melting Temperature: 55C to 65C

Similar or closely matched (within 5°C) to ensure that they will have the same hybridization kinetics during annealing

Melting Temperature: ___C to ___C

Similar or closely matched (within _°C) to ensure that they will have the same _______________________ during annealing

Primers having 3 or more dinucleotide repeats should be avoided

(e.g., ATATATAT)

The maximum number of repeats is 4 dinucleotides

Primers having _ or more dinucleotide repeats should be avoided

(e.g., ATATATAT)

The maximum number of repeats is _ dinucleotides

GC Clamp

Presence of G or C bases within the last 5 bases from the 3’ end helps promote specific binding More than 3 G’s or C’s should be avoided

Spaced 150-bp to 500-bp apart

Optimal Distance Between Primers

100-bp to 700-bp

Product Size

Self Dimer

Formed by intermolecular interactions between (same sense) primers where the primer is homologous to itself. A 3’end self dimer with G of -5kcal/mol is tolerated

Hairpins

Formed by intramolecular interaction within the primer. A 3’ end hairpin with G of -2 kcal/mol is tolerated

Cross Dimer

Sense and antisense

Gibbs free energy (ΔG) is the measure of the amount of work that can be extracted from a process operating at a constant pressure

Calculated using the nearest neighbor method:

Gibbs = Enthalpy - temp(entropy)

)________________ (ΔG) is the measure of the amount of work that can be extracted from a process operating at a constant pressure

Calculated using the nearest neighbor method:

Gibbs = ΔG = ΔH – TΔS

Enthalpy

is the amount of heat energy possessed by substances

entropy

the amount of disorder a system exhibits