Week 5 Nucleic Acid Based Methods

Question 1

What is the goal of PCR and when was it developed?

Answer 1

In 1983 and the goal was to yield sufficient DNA targets for use in subsequent gene analyses

Question 2

What are the 3 steps of PCR

Answer 2

1. Denaturation - at a high temperature to separate the double stranded DNA
2. Annealing of primers (both forward and reverse) at a cooler temperature
3. Extension - addition of nucleotides to generate new DNA fragments at a slightly higher temperature

Question 3

What is the typical workflow of an environmental molecular ecological study?

Answer 3

1. DNA extraction of the environmental samples
2. PCR of the target genes
3. Potential agarose gel electrophoresis to verify correct amplicons
4. Either differentiating genotypes or cloning

Question 4

Where are restriction nucleases produced?

Answer 4

They are produced by bacteria to protect them from viruses by degrading incoming viral DNA

Question 5

What do restriction nucleases do?

Answer 5

Each recognizes a specific sequnce of 4-8 nucleotides in DNA, and the cleaves the DNA at that site

Question 6

What is restriction fragment length polymorphism?

Answer 6

When restriction nucleases are applied to gDNA or PCR amplicons, it has become a popular genotyping method, with rapid determination of inter- and intra - species variations, often used on forensic testing

Question 7

This is cloning of small inserts, explain the steps in the picture

Answer 7

1. The foreign DNA and plasmid are cut using a restriction enzyme at a restriction site. In the plasmid it cuts through the metabolizing lactose gene
2. The creates sticky ends for the foreign DNA and cloning vector to anneal and ligase sticks them together
3. The bacteria are grown on a media containing both ampicillian and x-gal a chemical metabolized the same way as lactose. Plasmids lacking the foreign insert are able to metabolise the x-gal, turing blue. Bacteria without a plasmid is killed by the ampicillin

Question 8

What is a promoter region in cloning vectors?

Answer 8

Found before the site where the gene of interest is added, directs RNA polymerase to make copies of the gene of interest

Question 9

How can you get vectors inside a bacterial cell in cloning?

Answer 9

Heat shock or electrical shock as the cell membrane becomes more relaxed

Question 10

How can a hybrid gene be made with PCR?

Answer 10

A PCR with primer of front end of gene 1, of rear end of gene 2 and overlap the primer

Question 11

Give three examples of PCR in genetic engineering

Answer 11

1. Synthesis of hybrid gene using overlapping primers
2. Directed mutagenesis
3. Engineering deletion and insertions

Question 12

What is amplicon sequencing?

Answer 12

Sequencing of PCR products of a specific target gene in a mixture of sequences using next generation sequencing

Question 13

What is amplicon sequencing ideal for?

Answer 13

Diversity studies

Question 14

How does amplicon sequencing work?

Answer 14

It introduces a short index ‘barcode’ tag into each sample a normal PCR is carried out and the unique endings of each sequence is attached

Question 15

What is the disadvantage of amplicon sequencing

Answer 15

Once it is sequenced, no archives or amplicons are there for later use as controls

Question 16

What is eDNA

Answer 16

Extra-cellular DNA shed from an organisms into the environment

Question 17

What is eDNA being used for?

Answer 17

• Detection of rare species
• Invasive species to inform management actions

Question 18

What are the disadvantages of eDNA?

Answer 18

• Dont know how many species are there, just that they are present
• Specialist equipment
• Dont know if they are actually alive

Question 19

What is qPCR?

Answer 19

Real-time PCR

Question 20

What is the Ct in qPCR and what is its relationship?

Answer 20

It is the number of cycles at which amplification enter its exponential phase and it has a log-linear relationship with gene quantities (a high quantity of genes = a low ct)

Question 21

What are the two types of detection in qPCR

Answer 21

1. DNA binding dyes - bind to any double stranded DNA
2. Hybridisation probes - specific to gene of interest

Question 22

What is happening here?

Answer 22

SYBR green will bind to any double stranded DNA, therefore, a melt curve analysis is used to determine the melting temp of the PCR amplicon as a verification of product size. The melt point is where it drops off as you increase the temp you get lower fluorescence as it is no longer bonded in double helix

Question 23

What is the methods for qPCR with TaqMan probes?

Answer 23

1. There is a fluorophore and quncher attachedon the target DNA
2. When they are too close together no green light is emitted
3. When it is elongated the nuclease activity cite the probes, releasing the two molecules and thus fluorescence

Question 24

What is reverse transcriptase?

Answer 24

An enzyme used to generatre complementary DNA (cDNA) from a RNA template termed reverse transcription

Question 25

What is reverse transcription used for?

Answer 25

Replication of reteroviruses

Question 26

What are the four steps of reverse transcription?

Answer 26

1. Incubate the reverse transcriptase to synthesise the cDNA strand 2. When the cDNA is completed, hydrolyse the RNA strand 3. Incubate with DNA polymerase to synthesise the second DNA strand 4. Incubate with terminal transferase to add single-stranded tails

Question 27

How is reverse transcription different to transcription?

Answer 27

Reverse transcription is making ‘DNA’ from RNA

Question 28

What is the technique used for absolute quantification of PCR?

Answer 28

Real-time PCR

Question 29

How do you use a realtime PCR?

Answer 29

Compare the Ct value of a sample against a standard curve

Question 30

What is the equation for a reverse transcription real time PCR?

Answer 30

RT-qPCR = RT + real-time PCR

Question 31

Why is relative quantification needed in qPCR?

Answer 31

To confirm that reference gene has consistent expression in experiment

Question 32

How do you use endogenous control in qPCR?

Answer 32

1. Amplify target and reference genes 2. Compare Ct values for target and reference gene (difference in Ct) 3. Compare the sum of Ct between the control and test sample

Question 33

What does endogenous reference assume?

Answer 33

That all amplification efficiencies are identical

Question 34

What is the equation for endogenous references in qPCR?

Answer 34

Question 35

You should take multiple endogenous genes, which ones would you pick from here and why?

Answer 35

The green ones as they are the most stable

Question 36

Name 3 pros of real time PCR?

Answer 36

1. Sensitive 2. Large dynamic range 3. Robust 4. Once assay is built they are very quick 5. Can run multiplex reactions

Question 37

What are the cons of qPCR (name 4)

Answer 37

1. Cost 2. Optimisation of assay 3. Validation of endogenous references 4. Still requires mRNA to cDNA, efficiency considerations 5. Requires pre-selection of target genes 6. Requires sequence information for targets and references

Question 38

What are reporter gene assay and where do they go?

Answer 38

Genes that are used in genetic analysis because their products are easy to detect, such as antibiotic resistant genes and they normally go before or after the promoter region.