Practical applications of molecular biology

Question 1

what is PCR used for

Answer 1

PCR can be used to detect the presence or absence of specific genes
e.g. those encoding antibiotic resistance

Question 2

what is Dideoxy DNA sequencing or Sanger sequencing

Answer 2

1. requires 5 things: template DNA, DNA polymerase, primer, the four DNA nucleotides (dATP, dTTP, dCTP, dGTP), and Dideoxy versions (ddATP, ddTTP, ddCTP, ddGTP)
2. the Dideoxy versions of the nucleotides are added in very small conc.
3. mixture is amplified using PCR methods, when ddNTP is added to DNA chain during elongation the chain can’t grow longer (stops at ddNTP)
4. which ddNTP got added and how long the final chain is tells us what type of base and where it is in the DNA sequence e.g. ddATP got added and the chain was 5 bases long soooo the 5th base in the DNA sequence is Adenosine.

Question 3

what 4 components does PCR require

Answer 3

1. template DNA
2. DNA primers
3. DNA polymerase
4. deoxynucleoside triphosphates dNTPs

Question 4

what is Next-generation sequencing

Answer 4

• Principle is the same as dideoxy sequencing. But more sensitive, more targets, less time.
  1. no ddNTPs, just specialised dNTPs with fluorescent markers
  2. each time a dNTP is added a highresolution digital camera records the color of the
  fluorescence.
  3. Snapshot of each round of synthesis compiled to give DNA sequence

Question 5

what is Single-molecule real-time (SMRT) sequencing or PacBio

Answer 5

1. flow cell with thousands of tiny wells, each containing a single DNA polymerase, a single DNA template, and four fluorescently tagged dNTPs.
2. Initial binding of a nucleotide to the template generates a local fluorescent signal that disappears when the terminal phosphates are removed during incorporation of the nucleotide into the DNA.
3. SMRT sequencing enables very long fragments to be sequenced, up to 15-20Kb, or longer with very high accuracy.

Question 6

what is REVERSE TRANSCRIPTION POLYMERASE CHAINREACTION
(RT-PCR) and why is it used

Answer 6

1. it uses mRNA (e.g. from pathogens) called total mRNA to create complementary DNA (cDNA)
2. using reverse transcriptase enzyme and primers to create the cDNA
3. then cDNA gets amplified through PCR
   - it is used to sequence mRNA, create vaccines from pathogens with RNA, detect expressed/activated genes

Question 7

what is Quantitative RT-PCR and why is it used

Answer 7

1. Dyes added to the PCR fluoresce only when bound to double stranded DNA -
2. used to track the progress of the reaction and deduce starting conc. of the mRNA being amplified.
   - it is used to measure the expression of individual genes

Question 8

what is Next-generation sequencing (NGS) for RNA analysis and what is it used for

Answer 8

1. reverse transcriptase is used to copy all RNAs into cDNAs, which are then fragmented and
   sequenced by NGS methods
2. global analysis of mRNAs by
   RNA-seq provides a snapshot of gene expression.
3. More abundant RNAs will have more cDNA copies, resulting in higher numbers of “sequence reads” for those RNAs.
   - RNA-seq is used to identify and provides information about their relative abundance

Question 9

do RNA levels always match protein levels

Answer 9

no, because of post-transcriptional modifications

Question 10

how to purify proteins

Answer 10

1. lyse the cell: Add lysis buffer
   (eg detergents/lysosome)
2. centrifuge, then remove the cell debris at the bottom
3. add RNAse/ DNAse to degrade the RNA/DNA
4. precipitate with ammonium sulphate
5. Centrifuge and then resuspend in buffer and purify further…

Question 11

how does column chromatography work

Answer 11

1. sample applied to top of column filled with permeable gel matrix (e.g. cellulose)
2. Solvent passed slowly through and collected in separate tubes as it emerges
   from the bottom
3. Sample components travel at different rates through the column and are fractionated
   into different tubes

Question 12

what are the 3 types of matrices used for chromatography

Answer 12

1. ion-exchange chromatography - positively charged beads with negatively charged molecules bounded to it
2. gel filtration chromatography - porous beads, small molecules get delayed inside
3. affinity chromatography - beads with attached substrates that enzyme molecules attach to

Question 13

what is immunoprecipitation and why is it used

Answer 13

1. a rapid affinity chromatography method
2. Antibody-coated beads are added to protein extract in tube and mixed in suspension for a short period of time —thereby allowing the
   antibodies to bind the desired protein (i.e. no column)
   - used to purify small amounts of enzymes from cell extracts
   for analysis

Question 14

what does protein sequence and structure tell us about the protein

Answer 14

provide clues about protein
function

Question 15

what is SDS PolyacrylamideGel Electrophoresis (SDS-PAGE)

Answer 15

1. detergent sodium dodecyl sulfate (SDS) and the reducing agent Beta- mercaptoethanol are used to solubilise the proteins
2. Polypeptide chains form complex with SDS
3. SDS-protein complex (negative) migrates
4. Smaller are faster

Question 16

what is isoelectric focusing and what is it used for

Answer 16

1. At the isoelectric point, the protein has no net charge.
2. when a tube containing a fixed pH gradient is subjected to a strong electric field in the appropriate direction, each protein species migrates until it forms a sharp band at its
   isoelectric point.
   - Can be used to detect differences in [protein] e.g. stress, antibiotic treatment

Question 17

what is western blotting

Answer 17

1. Specific proteins can be detected by blotting with antibodies