Analysis of Nucleic Acids

Question 1

Define DNA cloning:

Answer 1

• selective amplification to generate homogenous DNA population:

Question 2

How does DNA cloning occur in vitro?

Answer 2

1. Cut DNA target sequence and replicon (sequence capable of independent replication) with same endonuclease so 2 DNA ends compatible
2. Purify + mix + join fragments with ligase
3. Transformation of recombinant DNA molecules into host cells
4. Selective propagation of individual cell colonies on agar plate (selective antibiotic resistance marker in replicon - only cells with replicon survive)
5. Expansion of cell culture and isolation of recombinant DNA

Question 3

What are restriction endonucleases?

Answer 3

• enzymes (dimers) that cleave DNA at specific recognition sequences
• usually 4-8 bp palindromic sequences (same forwards and backwards)
• can produce sticky ends (not straight cut with an overhang)
• will only cleave unmethylated DNA from invading organisms
• Longer recognition site occurs less frequently in DNA

Question 4

How does electropheresis work?

Answer 4

• DNA negative (backbone) moves towards anode (positive charge) when passed through electric current - forced to travel through porous gel matrix (agarose)

Question 5

What occurs after separation?

Answer 5

1. Isolate gel
2. Transfer to membrane to form a replica with probe detected by photographic film
   
   • Used in familial genetic analysis

Question 6

What is the function of southern blotting?

Answer 6

• transferring fragments onto membrane that immobilizes them so

Question 7

What are the steps in southern blotting?

Answer 7

1. Membrane washed with probes –> attach to complementary sequences
2. Once reannealed excess is washed
3. Expose to photographic film in dark to show fragment position

Question 8

Define hybridization:

Answer 8

homologous single-stranded DNA or RNA molecules combine via homologous base-pairing to form double-stranded molecules.

Question 9

What must one do for the probe to attach?

Answer 9

• denature DNA (make single stranded) in order for probe to bind to DNA (radioactive/fluorescent)

Question 10

What is a a hybridization assay?

Answer 10

• Target DNA immobilized on solid support (e.g nylon) which readily binds single stranded nucleic acids
• Hybridized with solution of labeled probe
• Use of photographic film or fluorescence

Question 11

What is hybridization stringency?

Answer 11

• how strictly a probe binds to DNA - power to distinguish between related sequences
• Low: can bind with degree of mismatch
• High: only if perfect match
• Measures nucleic acid duplex stability

Question 12

What does stringency increases with?

Answer 12

1. Increasing temperature
2. Decreasing sodium ion concentration (destabilizes DNA complex by neutralizing phosphate backbone)
   • Reaction conditions may dictate stringency
   • Also affected by strand length and GC ratio

Question 13

Define melting temperature:

Answer 13

• midpoint temperature of transition from double to single stranded nucleic acids
• in mammals ca. 87%
• Hybridization carried out 25 degrees below Tm

Question 14

What is PCR?

Answer 14

• in vitro amplification of specific target DNA sequences in heterogenous sequence mix

Question 15

What are the steps in PCR?

Answer 15

1. Denature - 94
2. Anneal - 50-60 primers anneal by lowering temperature - 2 primers, one complimentary to each DNA strand i.e one forward one reverse
3. Extend - 72 thermostable DNA polymerase - taq (thermophilus aquaticus)polymerase and dNTPs extend from 5’-3’ from primers
   
   • Repeated ca. 30 cycles

Question 16

How should a primer be designed?

Answer 16

• Length: 20 nucleotides gives required specificity
• Avoid tandem nucleotide repeats (multiple of same base e.g AAAA) - may form hairpins, percentage of GC and length should give an equal melting temperature for each primer
• Avoid complementary 3’ end pairs - may cause primer dimer

Question 17

What are the uses of PCR?

Answer 17

1. Detect point mutations
2. cDNA cloning
3. Gene expression - reverse transcription PCR
4. DNA sequencing
5. DNA microarrays - collection of microscopic DNA spots representing single gene robotically arrays on solid surface e.g glass slide

• Often used for expression profiling eg. monitoring simultaneous gene expression

Question 18

What is reverse hybridization?

Answer 18

• immobilized DNA/oligonucleotide probe and target DNA solution
• Collection of microscopic DNA representing single genes arrayed on solid surface
• Each oligonucleotide represent a single gene
• Isolate mRNA from types of cells to see pattern of binding of mRNA to oligonucleotides

Question 19

What is the function of microarrays?

Answer 19

• Used for expression profiling - monitor expression levels in thousand of genes simultaneously
• Utilizes selective nature of DNA-DNA or DNA-RNA