Chapter 5

Question 1

Deoxynucleotide

Answer 1

Made up of organic base, a ribose sugar and phosphate

Question 2

dNTPs

Answer 2

The building blocks for amplication

Question 3

DNA Polymerase

Answer 3

an enzyme that catalyzes the synthesis of DNA molecules from nucleoside triphosphates

Question 4

Taq DNA polymerase

Answer 4

Thermophilus aquaticus

has strong processivity, but lacks proofreading capability, thermal stable

Question 5

Polymerase Chain Reaction

Answer 5

The repeated serial reaction involving the use of oligonucleotide primers and DNA polymerase that is used to amplify a particular DNA sequence of interest

Question 6

Denaturation

Answer 6

The alteration of a protein shape through some form of external stress

Question 7

Annealing

Answer 7

recombin(DNA) in the double-stranded form following separation by heat

Question 8

Elongation

Answer 8

the synthesis of new strands by making a complete copy of the templates

Question 9

Extension

Answer 9

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

Question 10

Oligo Primer

Answer 10

initiates DNA amplification in a PCR

Question 11

Agarose Gel

Answer 11

A polysaccharide matrix that functions as a sort of sieve

Question 12

Gel electrophoresis

Answer 12

a technique used to separate DNA fragments according to their size

Question 13

Restriction Enzyme

Answer 13

An enzyme (a protein-endonuclease) that has the property to cut DNA at or near a specific recognition nucleotide sequence (restriction site)

Question 14

Blunt End

Answer 14

A straight cut of restriction enzymes generates blunt ends, where both strands terminate in a base pair

Question 15

Sticky end

Answer 15

when one end of DNA is longer than the other (typically 4nt) after restriction enzymes

Question 16

Restriction fragment length polymorphism

Answer 16

Detects the variation in length by a particular piece of DNA

Used to detect a single nucleotide substitution that leads to the disease

Question 17

Basic components in a PCR

Answer 17

• 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

Question 18

Thermocycler

Answer 18

• 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

Question 19

Restriction Enzymes and their function

Answer 19

Enzymes used to cut DNA. They bind to specific DNA sequences and then cleave the DNA at two defined locations, on on each strand

Question 20

Amplicons

Answer 20

amplified DNA fragments, called amplicons, are separated on an agarose gel using gel electrophoresis

Question 21

Friedreich Ataxia (FRDA)

Answer 21

• 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)

Question 22

Single Nucleotide Polymorphisms

Answer 22

(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

Question 23

Hereditary Hemochromatosis (HH)

Answer 23

• 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