DNA Analysis Techniques

Question 1

PCR

Answer 1

Polymerase chain reaction; no longer need to clone DNA in plasmid to amplify a gene; amplify any region of the genome using a very small amount of isolated genomic DNA and a set of primers

Question 2

PCR primers

Answer 2

short segment of single-stranded DNA that specify the region of amplification by providing a 3’ OH to DNA polymerase

Question 3

PCR steps

Answer 3

1) Denature (95C): high temperatures separate DNA strands by breaking hydrogen bonds between bases
2) Anneal (55C): lower temperatures allow primers to anneal to template DNA
3) Extend (70C): optimal temp for DNA pol to add nucleotides to synthesize new strand in 5’ to 3’ direction
Repeat (~35 cycles) to make millions of copies of DNA

Question 4

Agarose gel electrophoresis

Answer 4

sorts DNA segments by size; often use to confirm successful amplification and determine size of PCR product; used to visualize bands and estimate their size

Question 5

qPCR

Answer 5

Quantitative PCR; detected as amplification progresses by measuring an increase in fluorescence during the exponential phase because the signal is very strong; more sensitive and accurate than gel electrophoresis

Question 6

SYBR green

Answer 6

fluorescence molecule that binds to dsDNA

Question 7

Taqman probe

Answer 7

primer has fluorescence molecule (R) and quencher (Q) that suppresses fluorescence. Primer binds to ssDNA, when amplification occurs, DNA pol cleaves probe and releases fluorescence molecule from quencher so fluorescence is activated.

Question 8

Cq value

Answer 8

cycle number that fluorescence of a sample exceeds a pre-determined threshold set by experimenter; used to quantify amplification and is relative to amount of template DNA. Always run with multiple samples for comparison; lower Cq = more amplification = more gene expression because it hit the threshold earlier