Lecture 37

Question 1

What components are needed for a genetic construct to work?

Answer 1

Promoter, enhancers, start codon, transcription initiation site, stop codon

Question 2

What are the basics of gel electrophoresis?

Answer 2

DNA has a negative charge so it migrates toward the positive electrode; separation is based on fragment length; DNA is usually cut with restriction enzymes or is the product of PCR; gel is soaked in ethidium bromide which sticks to DS DNA and fluoresces when exposed to UV light

Question 3

How are plasmids designed for cloning?

Answer 3

They contain selectable markers that you can use to test to see if the bacteria has incorporated the plasmid

Question 4

How is LacZ used in cloning?

Answer 4

Forms a blue precipitate when a bacteria has it and thus bacteria will be blue when grown; if they don’t have it, the bacterial colonies will be white

Question 5

What are the basics of PCR?

Answer 5

Requires 5 chemical components (DNA template, DNA polymerase enzyme, primers, nucleotides, reaction buffer); DNA is heated to separate 2 DNA strands, quickly cooled to allow short primers to anneal to sequences, reheated and DNA polymerase synthesizes 2 new double-stranded molecules

Bottom Line: exponential amplification of target sequence

Question 6

What is Quantitative PCR?

Answer 6

Quantitatively determining the amount of DNA amplified as the reaction proceeds

Question 7

What is chromosome walking and chromosome jumping?

Answer 7

Chromosome Walking: sequencing fragments from a point near to a gene of interest

Chromosome Jumping: large fragment is circularized and the junction is sequenced

Question 8

What are restriction enzymes?

Answer 8

Recognize and cut DNA at specific nucleotide sequences

Question 9

What is a type II restriction enzyme?

Answer 9

Cut DNA at defined positions close to or within their recognition sequences; most useful restriction enzymes

Question 10

What are cohesive ends? Blunt ends?

Answer 10

Cohesive: fragments with short, single-stranded overhanging ends

Blunt: even-length ends from both single strands

Question 11

What is a probe?

Answer 11

DNA or RNA with a base sequence complementary to a sequence in the gene of interest and with a radioactive or chemiluminescent molecule attached allows visualization

Question 12

What probe would you use when doing a southern/northern/western blot?

Answer 12

Southern: DNA

Northern: RNA

Western: protein

Question 13

What is gene cloning?

Answer 13

Amplifying a specific piece of DNA via a bacterial cell

Question 14

What is a cloning vector?

Answer 14

Replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell

Question 15

What are linkers?

Answer 15

Synthetic DNA fragments containing restriction sites

Question 16

What is Taq polymerase?

Answer 16

DNA polymerase that is stable at boiling temperatures

Question 17

What is reverse transcriptase PCR (RT-PCR)?

Answer 17

RNA template first converted into a complementary DNA (cDNA) using reverse transcriptase; cDNA is then used as a template for exponential amplification using PCR

Question 18

What are some uses of PCR?

Answer 18

Detecting presence of viruses in blood samples, identify genetic variation, isolate DNA from ancient sources, amplify small amounts of DNA from crime scenes, introduce new sequences into a fragment of DNA

Question 19

What are some limitations of PCR?

Answer 19

Requires prior knowledge of at least part of the sequence of the target DNA, contamination a significant problem, Taq polymerase cannot proofread, size of fragment that can be amplified is usually 2000 bp

Question 20

What is a DNA library/cDNA library?

Answer 20

DNA Library: collection of clones containing all the DNA fragments from one source

cDNA Library: consisting only of those DNA sequences that are transcribed into mRNA