DNA Techniques

Question 1

gel electrophoresis

Answer 1

• first thing you do when doing blotting technique
• uses agarose gel to separate nucleic acids based on size –> current applied that causes negatively charged (phosphate groups) DNA molecules to move through gel towards positive electrode –> to view fragments, gel stained w/ ethidium bromide that inserts between nitrogenous bases and makes DNA visible under UV light
• confirms size but doesn’t tell identity of nucleotide sequence of fragments

Question 2

restriction fragment analysis

Answer 2

modification of gel electrophoresis
- restriction enzymes chop up DNA into fragments –> run fragments on gel –> then compare patterns in fragment generation between different individuals or species

Question 3

PCR (polymerase chain reaction)

Answer 3

amplification of specific region of DNA*
- can create billions of clones of DNA in hours
1. Denaturation: reaction mixture heated to denature (break H-bonds) between 2 DNA strands
2. Annealing (or hybridization): mixture is cooled to allow annealing (H-bonding) of short, single-stranded DNA primers complementary to opposite sides of both DNA strands
3. Extension: heat stable Taq DNA polymerase replicates the specific piece of DNA by adding nucleotides to 3’ end of primers, extending primers in 5’ -> 3’ direction
- cycle 1 = 2 DNA molecules, cycles 2 = 4 molecules, exponential

Question 4

colony blotting**

Answer 4

simple blotting technique + nucleic acid hybridization
many colonies on plate that has undergone blue/white screening –> colony blotting = finding gene of interest from 1000s of clones from DNA library
- blot DNA from plate/colony onto specialized membrane –> add labeled probe to membrane that’s specific to gene of interest –> allow it to hybridize –> then take image of resulting fluorescence

Question 5

nucleic acid hybridization

Answer 5

making complementary nucleic acid probe for sequence of interest –> then add something that will provide visual signal (probe labeled w/ tag that is radioactive or fluorescent)

Question 6

southern blotting**

Answer 6

for detection of specific DNA sequence
- combines gel electrophoresis of DNA fragments w/ nucleic acid hybridization
1. prepare chromosomal DNA samples and digest w/ restriction enzymes
2. separate DNA fragments by gel electrophoresis
3. blotting - DNA transfer: DNA is denatured (w/ alkaline solution) and transferred to a membrane/nitrocellulose filter –> resulting blot is exact replica of gel
4. nucleic acid hybridization: on blot, restriction fragment(s) w/ DNA of interest can be identified w/ a labeled nucleic acid probe that will hybridize w/ any complementary DNA fragments
5. probe detection: filter is then developed by autoradiography to detect DNA fragments that are complementary to probe
- southern blotting can detect normal and sickle-cell alleles of human B-globin gene
- simple summary of steps: 1. preparation of restriction fragments, 2. gel electrophoresis, 3. DNA transfer (blotting), 4. hybridization with radioactive probe, 5. probe detection

Question 7

DNA sequencing**

Answer 7

determines sequence of nucleotides in DNA molecule
- dideoxy ribonucleotide (or dideoxy-) chain termination method/Sanger method
- sequence 200-400 nts on average
- 4 separate reaction tubes set up
1. DNA is denatured and incubated with components (primer to get synthesis started, dNTP or deoxyribonucleotide phosphate to be used in DNA synthesis, ddNTP or dideoxyribonucleotide phosphate that will stop/terminate production of nucleotides, and DNA polymerase + DNA template strand mentioned in step) for sequencing reaction in small tube
2. synthesis of new strand in 5’-3’ direction until dideoxy (dd) nucleotide inserted at random instead of normal nucleotide –> produces series of labeled strand of varying length
3. labeled strands separated through gel in capillary tube (capillary electrophoresis) –> fluorescent detector detects labeled fragments indicating nucleotide at end of sequence –> sequence read (shortest to longest) and complementary to template strand

Question 8

“next generation sequencing” techniques**

Answer 8

• cheaper and faster
  1. Illumina sequencing by synthesis –> improvement on Sangar method –> detects fluorescently labeled nucleotide upon incorporation
  2. Ion proton sequencing (Ion torrent): based on detection of hydrogen ions released into growing DNA template during incorporation of new nucleotides
  3. Pyrosequencing - detects phosphate: adaptors added to ends of DNA fragments –> separated into single strands –> immobilized into beads (1 DNA strand per bead) –> PCR amplification –> sequence by pyrosequencing - measures pyrophosphate (PPi) released whenever a nt is incorporated
• more details in lect 6