Vikram & Tracey Lectures

Question 1

Define PCR.

Answer 1

A technique used to create several copies of a particular section of DNA.

Question 2

Outline the PCR process.

Answer 2

Denaturation; DNA is heated to 96C for 30 seconds to separate the strands.
Annealing; DNA called to 55C to allow short single strand primer to anneal to their complementary sequences.
Extension; DNA is heated again to 72C, DNA polymerase synthesis new DNA strands creating two new double-stranded DNA molecules.

Question 3

Define Taq Polymerase and how it relates to PCR.

Answer 3

Taq is a thermostable DNA polymerase used in extension, its derived from a thermophilic bacterium. (It’s able to withstand the heating processes of PCR)

Question 4

What is the difference between forward and reverse primers?

Answer 4

Forward; PCR primer which are complementary to antisense strand of DNA, amplifies this strand at the 5’ end of the PCR product.
Reverse; PCR primer which are complementary to sense strand of DNA, amplifies this strand at the 3’ end of the PCR product.

Question 5

How to detect, visualize, and optimize PCR?

Answer 5

Detection shows if the PCR has worked, this is done via gel electrophoresis; seepages DNA/sample according to size via small pored mech and charged ends.
Visualization of the gel is stained with fluorescent dye that binds to DNA which can then be seen under UV light.
Optimization; things can go wrong in PCR, for example, copies don’t amplify, contamination, etc…

Question 6

Define qPCR?

Answer 6

Real time PCR can monitor the amplification and quantification of your sample in real time. (Detection methods include DNA-binding dyes, SYBR Green I)

Question 7

What are the advantages to qPCR?

Answer 7

Accoutrements quantification between samples, small changes can be detected, no post PCR processing, etc…

Question 8

Define dPCR.

Answer 8

Digital PCR

Question 9

Compare qPCR and dPCR.

Answer 9

qPCR; references needed, no-post PCR processing, collected data in exponential growth phase, detection capable down to a 2-fold change.l
ddPCR; no references needed, minimal PCR processing, end-point PCR, linear response to number of copies.

Question 10

Define Ct.

Answer 10

Cycle number at which detectable signal is achieved.

Question 11

Define Tm.

Answer 11

The melting temperature of primers.

Question 12

Define deltadeltaCt.

Answer 12

Method of analysis of qPCR in which you determine the level of expression of the gene of interest.

Question 13

Define plateau phase of PCR reaction.

Answer 13

No detectable increase in the concentration of DNA.

Question 14

Define proteomics.

Answer 14

Study of the complete complement of proteins present in a cell to system.

Question 15

Outline ESI. (Draw)

Answer 15

ESI is a technique to generate ions for MS by applying high voltage to a liquid to procure an aerosol.
1. ESI induced a high voltage on peptides to atomize their particles into tiny charged droplets.
2. As solvent evaporates, the charged intensity of the droplets increases, causing it to split into charged ions.

Question 16

Outline Single MS and MS/MS(Draw).

Answer 16

Single; identifies m/z of peptides only, the peptide is then identied by comparison to proteomics database of predicted m/z of trypsinized proteins.

Tandem; - Tandem MS is based on coupling mass spectrometers together in a series to analyze complex mixtures. The method uses two mass filters arranged sequentially with a collision cell between them.
- Essentially, MS/MS pulls each peptide from the first MS, breaks the peptide bond, and identifies each fragment based on their individual mass to charge ratio (m/z).

Question 17

Outline MALDI. (Draw)

Answer 17

MALDI is soft ionization that involves a laser striking a matrix of small molecules to create a gas phase without decomposing the molecules.
The general process in which this is done is;
Ionization; this step ensures all molecules are charged
Analyses; separates the molecules by mass-charge ratio (m/z) - smaller molecules reach detector faster.
Detector; determines what the sample consists of.

Question 18

Outline PMF. (How does it differ from MS/MS?)

Answer 18

Peptide mass fingerprint (PMF) is an analytical technique used for protein identification in which an unknown protein of interest is cleaved into smaller pieces in order to determine the mass and therefore identify the protein from a proteomics database.

*PMF, unlike MS/MS, doesn’t sequence the peptide, instead we use database searches to give us sequence information of the peptide.

Question 19

Outline the two approaches used to analyze proteomes.

Answer 19

2D gel electrophoresis combined with MALDI; protein quantification is achieved by difference in gel electrophoresis.
Liquid chromatography using ESI; protein quantification achieved by; (1) label free using peptide intensities (2) labeling with stable isotope.

Question 20

Define/Outline bottom-up proteomics & top-down proteomics (Explain the difference). (Draw)

Answer 20

Bottom-up; starts with the protein in question but requires a step in digestion using trypsin, once completed - MS/MS is used to procure fragmented ions. (Is there a better way to word it, research?)
Top-down; starts with the protein in question goes through MS/MS to procure fragmented ions.

Question 21

What are the advantages and disadvantages of ESI?

Answer 21

Adv; simple way to ionize non-volatile solutions.
Dis; experimental/technical parameters need to be strict.

Question 22

What is the ‘work flow’ of the 2 approaches used to analyze proteomes?

Answer 22

Sample prep.; SDS page, 2D gel
Protein digestion; Trypsin
Ionization; ESI, MALDI
MS; TOF, Quad
Analysis; peptide search

Question 23

Outline/Draw the PMF ‘work flow’.

Answer 23

Protein separation; SDS page
Protein digestion; trypsin
MS; TOF, Quad
Digestion (again)
Comparison; peak analysis

Question 24

Describe why MS/MS is is preferred over peptide mass fingerprinting (PMF) for highly complex protein samples.

Answer 24

• The peptide mass fingerprinting approach is usually used for samples where the protein of interest can be purified quite well, because peptide ion signals from different proteins can interfere with each other in an individual mass spectrum which can reduce the specificity of the PMF.
• In MS/MS, individual peptide ion species are isolated in the mass spectrometer and are subjected to fragmentation. The masses of the peptides and their fragments are measured, making it more applicable to complex mixtures, because a large amount of information is obtained for each peptide.

Question 25

Describe the advantages and disadvantages of ESI and MALDI.

Answer 25

ESI -
Adv; simple way of ionising non-volatile substances
Dis; experimental/technical parameters are strict.

MALDI -
Adv; fast, accurate in identification
Dis; extremely expensive

Question 26

Outline 2 advantages of using proteomics compared to genomics.

Answer 26

Proteomics allows for a greater understanding of the complexity of biological systems and the process of evolution than the study of genetic code alone.
Through proteomics approach, researchers can detect more protein targets than through genomics, which leads to highly effective biomarker studies, clinical trials, and drug discovery.
(Same genome, different proteomes)

Question 27

What is chain termination sequencing or Sanger’s method?

Answer 27

Sangers method is a cheap technique used in labs, where a DNA/primer sample is split into 4 (G, A, T, C).
Each tube contains the sample, 4 normal deoxynucleotides (dATP, dCTP, etc), a DNA polymerase, and one appropriate dideoxynucleotide (e.g. ddATP) in limited quantity.

Question 28

What is shotgun sequencing?

Answer 28

This method involved randomly breaking up the genome into small DNA fragments(enzymatic approaches) that are sequencing individually. Then assemble the overlapping DNA sequencing(rmb workshop). Final, assembled sequence.

Question 29

What is primer walking?

Answer 29

Another way of obtaining more length in the Sanger sequencing approach is primer walking.

Question 30

Define coverage.

Answer 30

Coverage is number of times a base is covered or read

Question 31

The evolution of next generation sequencing (NGS)?

Answer 31

When creating HGP using Sanger sequencing, it was found to be inefficient hence leading to NGS, where:
Second generation; clonal amplification of DNA templates on a soldi support matrix.
Third generation; PCR-free protocols.

Question 32

What is the difference in deoxynucleotides and dideoxy?

Answer 32

When a deoxynucletide is added to the 3’ end, chain continues.
When dideoxy is added, chain terminates.

Question 33

What is the manual and automated process in Sanger sequencing?

Answer 33

The manual approach to sequencing involves radio labelled or fluorescently labelled oligonucleotides(ddNTPs). Gel is exposed to UV to see.
Nowadays, the automated process is used: where the dideoxys’ are fluorescently labelled in different colors, a much simpler process.

Question 34

What is the difference between whole genome sequencing, whole exome sequencing, and targeted sequencing?

Answer 34

Whole genome; the process of determining the entirety of the DNA sequence of an organisms genome.
Exome; used for sequencing all of the protein coding regions of genes in a genome. (Cheaper, not so much data)
Targeted; focuses on specific genes. (When you’re interest at only specific genes instead of whole) (most cost effective one)