Analyzing the Cell II

Question 1

Single Nucleotide Polymorphisms

Answer 1

SNP variants can be neutral, pathogenic, or predisposing

Question 2

Restriction Endonucleases

Answer 2

Enzymes isolated from bacteria that cut DNA at specific sites

Question 3

Agarose Gel Electrophoresis

Answer 3

Different than SDS-PAGE; DNA is already charged

Question 4

Ligase Reaction

Answer 4

Much easier with compatible cohesive ends

Question 5

Cloning Vectors

Answer 5

Plasmids have been engineered to serve various functions; carry and replicate manipulated gene products

Question 6

DNA Libraries

Answer 6

Every gene in the human body can be put into bacteria, stored indefinitely, and propagated at anytime; how we discovered most genes; includes introns and exons

Question 7

cDNA

Answer 7

DNA copy of mRNA; no introns, much smaller than original gene, requires viral enzyme (reverse transcriptase)

Question 8

Polymerase Chain Reaction (PCR)

Answer 8

Amplify isolated DNA regions; primers are starting points for replication (complimentary to some portion of single stranded DNA)

Question 9

Purpose of PCR

Answer 9

Earlier detection of microorganisms (HIV, bacterial/fungal infections); detection of specific genetic mutations (cystic fibrosis, thalassemia, hereditary hemochromatosis, Huntington’s, Fragile X)

Question 10

Steps to PCR

Answer 10

1. Double-stranded DNA obtained from a patient/pathogen
2. Subjected to high temps to denature to ssDNA
3. Primers designed to complement sequences that flank each end of DNA in 3’-5’ direction
4. Allowed to anneal
5. Add Deoxy Nucleotide Triphosphates (all four)
6. Taq Polymerase synthesizes copy of DNA by extending the primers on both ends; DNA doubles and becomes amplified each cycle

Question 11

Taq Polymerase

Answer 11

Synthesizes copy of DNA by extending the primers on both ends during PCR

Question 12

Advantage and Disadvantage of PCR

Answer 12

Advantage: Very small amount of template DNA needed; 10^9 fold amplification from trace amount of DNA

Disadvantage: Need to know the sequence of the flanking DNA for primer design, error prone, amplification of contaminating DNA

Question 13

Quantitative PCR (qPCR)

Answer 13

Used to quantify copy number of a specific gene in two or more samples in real time; along with primers, includes a probe which fluoresces only in presence of the PCR product; used to detect levels of an infectious agent or determine levels of gene expression

Question 14

Restriction Fragment Length Polymorphisms (RFLP)

Answer 14

Genomes from 2 individuals cleaved with a set of restriction enzymes and the fragments resolved on a gel, the pattern is different; DNA fingerprinting; used in forensic analysis, paternity testing, and disease detection

Question 15

Variable Number of Tandem Repeats (VNTR)

Answer 15

Pattern of short tandem repeats (STR) occurs in genome but varies in individuals; VNTR repeat regions isolated from genomic samples by flanking restriction sites or through PCR; useful in identification and severity of inherited diseases (Huntington’s, Fragile X, Frederich Ataxia)

Question 16

DNA Microarrays

Answer 16

Used to determine overall change in gene expression between 2 samples

Question 17

Steps in DNA Microarrays

Answer 17

1. Isolate RNA from sample, make cDNA using reverse transcriptase, and add a fluorescent dye (called a probe)
2. Add different fluorescent dye to the second sample
3. Inject probe into the chip, it will bind with its complementary DNA sequence
4. Scanner will measure intensity of each spot and calculate fold-difference in gene expression

More intense the signal, higher degree of binding of probe, higher level of gene expression