Epigenetics and DNA technology

Question 1

What is epigenetics

Answer 1

Study of changes in organisms due to modification of gene expression rather than alteration of the genetic code itself
-epigenome remains flexible as environmental conditions continue to change

Question 2

Recombinant DNA technology

Answer 2

-laboratory manipulation of DNA in which DNA fragments from various sources are severed, combined, and reinserted unto living organisms

Question 3

Restriction enzymes

Answer 3

(Scissors)

• naturally occurring in bacteria
• use a palindromic cut
• blunt end cut
• sticky end cut (overhang)
• unpaired bases in sticky end can be used to join 2 fragments of DNA together (initially held by HB)
• DNA ligase is used to complete the fusion by forming phosphodiester bonds

Question 4

Plasmids

Answer 4

-small circular pieces of DNA found in bacteria
-plasmid is cut w same restriction enzyme as target gene segment and placed in solution
-sticky ends connect
-bacteria now expresses inserted gene
(Like insulin)

Question 5

Gel Electrophoresis

Answer 5

(Strainer/ barcode)

• a process used to separate DNA fragments based on their relative lengths
• DNA is treated w restriction enzymes to cut into different sizes
• Then it’s put into wells
• DNA has a negative charge because of phosphate groups so when the current is on DNA will move towards the positive electrode
• small fragments will be able to move through the gel faster than large ones
• this allows researchers to compare sizes of the fragments from an unknown sample to that of a known sample

Question 6

Polymerase Chain Reaction (PCR)

Answer 6

(Photocopier)

• used to make multiple copies of a DNA fragment
• begins with solution of DNA fragments, DNA primers, Taq polymerase, and free nucleotides
• heat is used to separate 2 strands of DNA
• the mixture is cooled and the DNA primers will anneal to strand of DNA
• Taq polymerase starts at primers and builds the complimentary strands using free nucleotides
• the process repeats and DNA is replicated in an exponential fashion

Question 7

What mixture is used in PCR

Answer 7

DNA fragments
DNA primers
Taq polymerase
Free nucleotides

Question 8

Why is sequencing DNA valuable?

Answer 8

• identify mutations
• locate regulatory sequences
• specific gene reference

Question 9

Describe the Sanger Sequencing Method

Chain termination

Answer 9

-relies on addition of a labelled dideoxynucleotide (ddNTP) to a growing DNA strand
-addition prevents other nucleotides to be added since there’s no hydroxyl group on the 3’ sugar)
-samples are separated using gel electrophoresis
1) PCR is used to copy the strand
2) 4 test tubes are prepared with:
•DNA polymerase
•DNA primer
•DNA strands
•normal nucleotides
•one type of labelled dideoxynucleotides (A, C, G, or T)
-DNA replication begins
-nucleotides are added to building strand until dideoxy is added
-fragments of varying lengths are generated, each ending in a labeled dideoxy nucleotide
-fragments from each test tube are loaded into well of gel Electrophoresis and separated based on length!