Lecture 1 - NA Isolation & PCR

Question 1

What is rDNA?

Answer 1

Recombinant DNA - artificially created, genetic sequences not usually found together in nature

Question 2

What are the 6 steps in molecular cloning?

Answer 2

1. Isolation of target gene
2. Insertion into vector DNA
3. Transfer into microbes via transformation/infection
4. Selection of microbial cells containing desired recombinant vectors
5. Growth of transformed microorganisms
6. Expression of the gene to obtain product

Question 3

Describe the Central Dogma

Answer 3

DS DNA
transcription
mRNA copy of one strand
translation
Amino acid sequence of protein

Question 4

What are the aims when isolating target nucleic acid?

Answer 4

High quantity, quality and integrity

Question 5

What are the 3 steps of isolating the target nucleic acid?

Answer 5

1. Disruption of cell membrane/cell wall
2. Separation and precipitation of nucleic acid
3. Dissolving nucleic acid

Question 6

What is cell lysis?

Answer 6

Outer boundary of cell membrane is broken down to release inter-cellular materials (e.g. DNA, RNA, protein or organelles)

Question 7

What are the 2 types of cell lysis methods?

Answer 7

Mechanical and non-mechanical

Question 8

What influences the lysis method chosen?

Answer 8

1. Ease of purification steps
2. Target molecules for analysis
3. Quality of final product

Question 9

In mechanical lysis, cell membrane is physically broken down using shear force. What are the 2 types of mechanical lysis?

Answer 9

1. High pressure homogeniser - cells in media forced through orifice valve using high pressure, disruption of membrane occurs due to compression and expansion
2. Bead milling - cells disrupted by tiny beads at high speeds

Very efficient method to lyse a wide range of cells
Limitation: Heating, degradation of cellular products and higher cost

Question 10

Non-mechanical lysis

What are the physical methods?

Answer 10

1. Freeze thaw cell lysis - ice helps break down cell membrane (can be time consuming, cannot be used for temperature sensitive cellular components)
2. Ultrasonic cavitation - shockwave disintegrates cell membranes, disruption is independent of biomass concentration (but large amount of heat generated)
3. Osmotic shock

Question 11

Non-mechanical lysis

What are the chemical methods?

Answer 11

1. Alkaline lysis - OH- ion in NaOH breaks fatty acid-glycerol ester bonds in cell membrane, sodium dodecyl sulphate (SDS) solubilises the proteins and membrane (mostly used for isolating plasmid DNA from bacteria)
2. Detergent lysis - disrupt hydrophobic-hydrophilic interactions of cell membranes, breaking the structure of water and making it less hydrophilic (chaotropic agents e.g. urea, guanidine and EDTA can also be used)

Question 12

Non-mechanical lysis

What is biological cell lysis?

Answer 12

Enzymes such as lysozyme, lysostaphin, zymolase, cellulase, protease, or glycanase are used

Highly specific

Lysozyme are used for bacterial cell lysis by specifically hydrolysing glycosidic bonds in peptidoglycan layers

Chitinases for yeast cell lysis and pectinases are used for plant cell lysis

Question 13

What are the methods of isolating nucleic acid?

Answer 13

1. TRIzol reagent (covered in practical class)
2. Silica-based extraction
3. Magnetic beads
4. Caesium chloride method

Question 14

Methods of isolating nucleic acid

What is silica-based extraction?

Answer 14

Negatively charged nucleic acids interact with positively charged silica membrane and are retained

Rest of the unbound soluble material filters through membrane

Impurities and loosely bound molecules are removes using washing buffer

DNA bound is eluted by dissolving in buffer and collecting

Question 15

Methods of isolating nucleic acid

How are magnetic beads used?

Answer 15

Positively charged magnetic beads entrap negatively charged DNA/RNA

DNA/RNA loaded beads are concentrated and separated from solution using a magnetic field

DNA/RNA can then be precipitated using ethanol

Question 16

Methods of isolating nucleic acid

How is caesium chloride used?

Answer 16

Molecular weight is used to extract DNA/RNA

Density gradient of CsCl is established in a centrifuge tube

Nucleic acid settles down as a band in the gradient where density of DNA and gradient is the same

Question 17

What to do if gene sequence of gene of interest is not available?

Answer 17

1. Construction of genome/cDNA library

2. Screening of gene of interest such as protein product, enzyme function

Question 18

What to do if gene sequence of gene of interest is available?

Answer 18

PCR amplification of target gene

Question 19

How is genome library constructed?

Answer 19

Genomic DNA isolated digested by restriction enzymes

Vector plasmid digested with restriction enzyme

Digested genomic DNA and vector are ligated together and transformed into bacterial host cells

Recombinant plasmid generated is subsequently transformed into a host cell for amplification

Question 20

How to generate cDNA from eukaryotic genes?

Answer 20

Reverse transcription of mRNA (to obtain functional sequence of gene)

Use oligo(dT) primer for polyA tail, and reverse transcriptase enzyme

RNA strand then hydrolysed by reverse transcriptase enzyme

Question 21

What are the 3 steps in PCR?

Answer 21

1. Denaturation - high temperature (90-100°C), separation of double DNA helix into two strands
2. Annealing - quick cooling (50-65°C), two primers bind to their complementary sequences on the template DNA strands
3. Extension - the reaction mixture is heated again (60-70°C), DNA polymerase extend the primers by adding complementary dNTPs

Question 22

How is PCR used in infectious disease diagnostics?

Answer 22

RNA extracted from swab samples using TRIzol reagent

RNA transcribed into cDNA and specific regions of interest amplified by PCR

Primers used target the sequences coding for RNA dependent RNA polymerase (RdRP), Spike protein (S), Envelope protein (E) and Nucleocapsid protein (N)

Question 23

How is PCR used in environmental science?

Answer 23

Environmental DNA (eDNA) metabarcoding is a method of assessing biodiversity

DNA from samples are taken from the environment via water, sediment or air

Primers used generally target the genes such as Cytochrome oxidase-I (COI), Ribulose biphosphate carboxylase large chain (rcbL) and 12s or 16s ribosomal RNA

Question 24

How is PCR used in forensic science?

Answer 24

The number of repeated nucleotides in hypervariable microsatellite sequence (VNTR - variable number of tandem repeat) is used to distinguish whether sample at the crime scene matches that of the suspect

Primers designed to prime at specific VNTR loci in homologous chromosomes