Molecular Techniques - Lecture 11 & 12

Question 1

In answering a molecular biology question or testing a hypothesis (related to genes); what major things do you consider?

Answer 1

The system (organism) to use
Molecular Technique to use
Gene that is involved

Question 2

Differentiate between phenotype vs gene directed analysis of genes

Answer 2

In phenotype, an observable trait/characteristic (after mutation) is used to identify the gene involved BUT In gene analysis, a gene of interest is isolated and disrupted in a model to observe changes in phenotype (if any)

Question 3

There are different kinds of DNA mutations that manifest at protein level. What are 5 types of small point mutations and what could be a bigger mutation?

Answer 3

Point mutations include 1. Misense (change in amino acid); 2. Nonsense (stop codon) 3. Silent (no change - same amino acid) 4. deletions 5. Insertions
Bigger changes are at chromosomal level

Question 4

What is the end product of mitosis and meiosis and explain using diploid and haploid cells and their differences. Use humans chromosomes to explain

Answer 4

Mitosis = 2 daughter cells with diploid chromosome (i.e 46 chromosomes - 23 from each parent in one cell) ALL SOMATIC CELLS ARE DIPLOID = 2n

Meiosis = 4 daughter cells with haploid chromosomes (I.e. 23 in each cell from each parent) GERM CELLS ARE HAPLOID = n

Fertilisation then occurs within germ cells (sperm and egg) - haploid, to produce a zygote which is diploid.

Question 5

In gene to phenotype analysis, highlight 3 techniques that are used and an essential material used in these techniques

Answer 5

Gene Knockout
RNAi
CRISPR (recent gene editing tech)
All of these techniques utilise Plasmid and Cloning Vectors to target genes!

Question 6

To evaluate the effect (whether positive or negative) of a successful knockout or gene editing, what sort of markers can be used?

Answer 6

Auxotrophs can be used as a marker
Fluorescence proteins or probes
PCR primers

Question 7

What are auxotrophs? Example?

Answer 7

Organisms that have have been manipulated so they lack a compound required for their growth and have to get this compound from their environment

Question 8

How can we text that gene knockout was successful at the locus of interest? Explain!

Answer 8

Easy: PCR (Primers can be designed to amplify regions flanking the target gene, and the absence of an amplicon would indicate a successful knockout)
Medium: Southern blotting (DNA sequences specific to gene of interest are detected/or not)
Further: Genome Sequencing

Question 9

What are 3 main characteristics features of ALL plasmids in molecular analysis and what are they used for?

Answer 9

Plasmids have: 1) An origin of replication
2) A selectable marker
3) Mechanism/Site where DNA can be inserted i.e. multicloning site
They are used as vectors (transporters of sequences into a foreign genome)

Question 10

Why do plasmid vectors need an OriC (Origin origin of replication)

Answer 10

The ori is a specific sequence of DNA that is recognized by the host cell’s replication machinery, allowing vectors to replicate the inserted gene/sequences are maintained in the host cell

Question 11

Restriction enzymes are palindromic, what does this mean and why is it important?

Answer 11

Palindromic means that the 5’ to 3’ and 3’ to 5’ on DNA strands are the same when read in the same direction. This is key because most restriction sites recognized by REs are palindromic

Question 12

Using the basic principle of Cloning, create a clone of Plasmid 1: tetracycline resistant AND Plasmid 2: ampicillin resistant. How would you test for successful cloning?

Answer 12

Digest both plasmids with REs»>Ligate with ligase both plasmids»>induce replication/growth»>select for recombined plasmid (successful cloning i.e. RARE recombination at some frequency). Grow Plasmid in the presence of markers=tetracycline and ampicillin and SELECT for Recombined Plasmids

Question 13

***What are the three possible outcomes when cloning a gene of interest (GOI) OR insert to a vector

Answer 13

-Plasmid could self-ligate
-GOI/Insert could self ligate as well
-Insert could ligate into target vector (RARE) Recomb

Question 14

Describe the cloning of a gene of interest into a plasmid vector with 2 selectable markers, how would you test/select for successful transformation ?

Answer 14

Isolate GOI and Plasmid»>Digest selectable marker plasmid & digest GOI with REs»>Ligate both»>select for recombined plasmid (successful cloning i.e. RARE recombination at some frequency). Grow Plasmid in the presence of markers=tetracycline and ampicillin and marker sensitivity and SELECT for Recombined Plasmids
(ANSWER NEEDS RETOUCHING)

Question 15

A gene/DNA clone is a new copy of a gene or DNA of interest?

Answer 15

No a clone is usually the same as its original DNA/gene sequence but it is isolated and inserted into a system that is easy to manipulate (Plasmid)

Question 16

Why do tags (like affinity or fluorescent tags) have to be added to plasmid vectors and what is their application?

Answer 16

Plasmid vectors are able to transcribe their gene or DNA of interest into protein; hence by adding tags when these proteins are expressed they can be used for detection/purification of proteins