Laboratory Techniques & Theory

Question 1

DNA Cloning

Answer 1

DNA Cloning: Is the propagation of a piece of DNA
Recombinant DNA: DNA molecule formed in the lab by joining together DNA seq. from different biological sources
-is an artificial creation - not found in nature

Question 2

How to Clone a Fragment

Answer 2

1. DNA for cloning purified from cells/tissues
2. Restriction enzymes used to produce specific DNA frag. - cuts at specific sequence
3. Frag. joined to other DNA molecules that serve as vectors
4. Recombinant DNA (frag + vector) transferred to host cell - molecule replicates producing clones
5. As host replicates, so does DNA molecule - creates pop. of cell carrying molecule
6. Cloned DNA can be recovered from host cell, purified & analysed
7. Cloned DNA can be transcribed, mRNA translated & encoded gene product isolated & used for research or sold

Question 3

Point of a Vector for DNA Cloning

Answer 3

Vector: carrier DNA molecules that transfer and help replicate inserted DNA fragments.
Vectors;
-allows fragment to be propagated (w/out vector it would be dissolved)
-Supplies a Origin of Replication (required for replication - occurs infrequently)
-Provides a selectable resistance gene - only about 3% of cells take insert (DNA frag) - need a way to tell which ones haven’t taken it up
-Selectable resistance gene allows us to select for cells that have taken it up (i.e. ampicillin resistance gene in E. coli - grow bacteria on ampicillin agar & only successful ones grow)

Question 4

Restriction Enzymes

Answer 4

• Produced by bacteria as a defensive mechanism against infection by viruses (can prevent viral infection by degrading DNA of virus)
• binds to DNA & recognises a specific nucleotide seq (RECOGNITION SEQUENCE)
  
  • usu. 4 to 6 nucleotides long (can be 8+)
  • can create a restriction fragment
• Most recognition seq. exhibit a form of symmetry = Palindrome (reads same both ways when read from 5’ to 3’)
• Sticky end: cut where a nucleotide is overhanging
• Blunt end: cut where nucleotides are flush/flat

Question 5

pUC18

Answer 5

• is a small vector (therefore can carry large DNA inserts)
• has original of replication
• large no. of restriction enzyme recognition seq. - clustered into one region called the polylinker site
• Recombinant pUC18 plasmids easily identifiable
• Has recognition site in middle of lacZ gene (gene produces beta-galactosidase, which when grown on X-gal agar produces a blue product)
  
  • if DNA fragment inserted anywhere into polylinker site, lacZ disrupted and becomes inactive - forms white colonies when grown on X-gal medium.

Question 6

Electrophoresis

Answer 6

-Separations of molecules on the basis of size and electrical charge in an electric field
-many types but gel electrophoresis used for DNA
How it works;
-P on DNA backbone = negative charge
-when current applied, DNA migrates to positive pole
-DNA moves through a gel matrix (of agarose) that forms a network of long polysaccharide chains w/ gaps between chains
-DNA pulled through gaps by electric field
-DNA mixed w/ substance that makes it heavy and sink to bottom of well
*Migrates according to size (inversely)

Question 7

What must be included in Gel electrophoresis

Answer 7

• Must have fragments of known sizes running;
  
  • is basically a control - shows DNA has run properly & provides a standard for comparison
  • Can work out a standard curve and estimate size of fragments based on how far they’ve travelled.

Question 8

Applications of Electrophoresis

Answer 8

• Look at extracted DNA quality (if no band on gel = DNA completely degraded)
• Enzyme reaction on DNA - might want to test the success of the reaction
• Test for mutations (e.g. breat cancer
  
  • mutations can change DNA (i.e. may cleave it or make it larger - can tell these things)
• Used to test for different allelic forms of a gene

Question 9

Gel Concentrations

Answer 9

-Gel can effectively separate 200 - 20 000bp

• 200bp = 3% agarose (over this amount it becomes too brittle)
• 20 000bp = 0.5% agarose (too liquid if under this amount

*Both extremes make it hard to get results

Question 10

Visualisation of Bands in Gel electrophoresis achieved by: (2)

Answer 10

• Staining gel w/ dye for specific nucleic acids (i.e. ethidium bromide) - fluoresces when exposed to UV light
• Can add label to DNA before placed in gel - i.e. chemical lable that can be dtected by adding antibodies

Question 11

Southern Blotting

Answer 11

Used when;

• larger DNA has a larger no. of bands that are indistinguishable in gel.
• Used to identify specific gene in large mess
• technique to transfer denatured, s.s, fragments
• membrane placed in a hybridisation soln of a labelled probe - probe binds to only part of DNA fragment
• Membrane then washed to remove any unbound probe

*RNA can be transferred to solid support from gel by Northern blotting (can show its size, its abundance or tissues

Question 12

Probes (as used to identify a specific gene in gel electrophoresis)

Answer 12

Probe: DNA/RNA molecule w/ base sequence complementary to a sequence in gene of interest

• cut DNA into frag. using 1 or more restriction enzymes, then separates fragments w/ gel electrophoresis
• separated fragments must be denatures & transferred to a permanent solid medium (via Southern blotting)

Question 13

Polymerase Chain Reaction

Answer 13

• method for amplification of a specific DNA sequence (200-400bp) without having to clone the DNA
• larger region for amplification is - less efficient process will be

Question 14

How PCR works

-DNA polyermase used

Answer 14

• start out w/ dsDNA
• heat @ 94 degrees for 3 mins to separate strands (denatures DNA)
• reduce temp to allow nucleotide primers to anneal to strands (65-70 degrees C) [anneals by H bonding]
  
  • primers of 15-20 bases used
• add DNA polymerase - shift temp to 72 degrees for 1 min to allow DNA polymerase to synthesis new strands
  
  • ALWAYS syn. in 5’ to 3’ direction
• heat to 94 degrees for 1 min and reapeat 30 - 40 times

-DNA polymerase from Thermus aquitirus (Taq polymerase) - can w/stand hight temps

Question 15

Primers in PCR

Answer 15

-Primers determine the specificity of PCR (tells you what sequences you’re going to amplify)
HOW?
-determined by the annealing temperatures which is set so that the primers only bind to perfectly complimentary sequence
-sometimes primers can bind to a similar sequence (has a mismatch)
-if use a temperature just below the Tm (melting temp) for PCR, all the mismatched DNA will be denatured and therefore won’t be replicated (as one mismatch in sequence decreases the Tm by about 5 degrees)

*this concept ensures that only one site (the right site) is amplified

Question 16

Ways to measure the amount of DNA produced via PCR

Answer 16

-use a dye that binds specifically to dsDNA and fluoresces (fluorescence should double after every cycle)

Question 17

3 Stages in PCR cycle

Answer 17

• 3 phases: growth, exponential & plateau
• when cureve starts to rise (going from growth to exponential) = Ct value
  
  • related to cells per mL (lower no. of cells = higher Ct Value - takes longer for curve to rise.

-Plateau = could be due to runnng out of reagents or the effect of repeated heating and cooling of DNA polymerase (can damage it)

Question 18

Traditional method of sequencing DNA

Answer 18

DNA -> break into fragments -> clone fragments -> extract DNA from colonies -> sequence each fragment/clone

• need big infrastructure to do this - one person working alone isn’t enough
  
  • large machines picking off white colonies (19 000/day)
  • need to incubate cells to allow them to grow
• Was beyond resources of individual research groups, although human genome was produced using this method

Question 19

Problem with tradition DNA sequencing to find answers to questions

Answer 19

-Having the human genome doesn’t give us answers
To use sequencing to find answers (e.g. of process);
-trying to find mutation that controls huntington’s disease:
-need 100 genomes of ppl w/ disease and 100 w/out disease to take into account natural variations w/n groups
-then need to compare the groups to figure out mutation

-In this hypothetical example, need at least 200 human genomes - would require a lot of manpower & time

Question 20

Process of Next Generation Sequencing

Answer 20

Genomic DNA -> Break -> separation technology -> sequence each fragment

• Beads have DNA molecules attached to them - all of the same copy
• Each reaction contains a different template
• DNA templates are separated into individual reactions and each reaction can be monitored individually

Question 21

Advantages of Not cloning DNA fragments

Answer 21

1. Some fragments are not clonable (i.e. might have v. strong promoter - will soak up all RNA molecule)
   
   • leaves gaps in genome
   • Next gen sequencing relies on physical separation (not biological), therefore all segments will be copied
2. Saves time - don’t need to grow things on colonies and wait

Question 22

The sequencing reaction (Next Gen)

Answer 22

• DNA polymerase adds bases to primer based on template strand = complementary strand
• If base added to primer, light is emitted, then next based added (due to being florescently labelled)
• By monitoring the emission of light as each base is added, we can determine the sequence of the template

Question 23

How Next Gen. sequencing has changed things

Answer 23

• Power of this method lies in its ability to set up parallel reactions - has resulted in massive decrease in cost
  
  • technology to carry this out is size of library photocopier (instead of rooms of machines)
• Has become possible for individual researchers to sequence genomes

Question 24

What we use Next Generation technology for

Answer 24

• Look at gene expression -useful for comparing expression in different tissues & in cancer cells
• Can use it to look at ecological studies - relationships between species OR comparisons (i.e. microbes found in healthy and unhealthy individual)

Question 25

Is traditional sequencing methods redundant??

Answer 25

- Not necessarily - Next generation sequencing does 50 bp reads - gives us a very large number of very small sequences - Hard to assemble genome from such small fragments - Using genome sequence from traditional methods can give us a sequence to model to (i.e. looking off a puzzle box)