Molecular Techniques

Question 1

What is nucleic acid hybridisation?

Answer 1

The formation of a stable duplex between two complementary strands of nucleic acid by hydrogen bonding

Question 2

What is DNA cloning?

Answer 2

A molecuar biology technique that creates many identical copies of DNA

Question 3

When was the beta-2 adrenergic receptor cloned?

Answer 3

1986
Since this, the beta-2 adrenergic receptor has been cloned, mutated, and recombinantly expressed such that many structural features involved in receptor function have now been defined.

Question 4

What are the different levels of structure of proteins?

Answer 4

A protein’s primary structure is defined as the amino acid sequence of its polypeptide chain
Secondary= local spatial arrangement of a polypeptide’s backbone
Tertiary= the 3D structure of an entire polypeptide chain
Quaternary= the 3D arrangement of subunits in a multi-subunit protein

Question 5

What can be done to a receptor to understand its function better?

Answer 5

DNA sequence can tell primary and other structures of the receptor
The receptors can be expressed in different contexts
Mutations can be introduced to examine structure-function relationships
Receptor can be over or under-expressed or removed from a tissue to workout what the receptor does at a tissue level

Question 6

What is in situ hybridisation?

Answer 6

It is a technique that allows for precise localisation of a specific segment of nucleic acid within a histologic section

Question 7

What is in situ hybridisation used for and what is an example?

Answer 7

It can provide information on cell type specific gene expression in a tissue
In situ hybridisation has been used to detect where the different alpha GABAa sub-units in a mouse cerebellum- alpha 1 receptors were only found in the purkinje cells but alpha 6 is much more varied.
This was useful as benzodiazepines only bind to alpha-1 receptors and thus could tell where a drug would act

Question 8

How does in situ hybridisation work?

Answer 8

Nucleic acids if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached (usually radioactive or fluorescent)
The probe is localised and quantified in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry
Old technique but still used

Question 9

What is RNA sequencing?

Answer 9

Generates lots of sequences simultaneously, RNA is extracted and DNA created by reverse transcriptase.
This DNA (complementary DNA) is sequenced using different sequencing platorms such as Illumina

Question 10

What is reverse transcriptase?

Answer 10

An enzyme that converts RNA to DNA

Question 11

What is PCR?

Answer 11

Polymerase chain reaction- amplifies DNA

Question 12

What are the steps in PCR?

Answer 12

Sample is heated so the DNA denatures into two seperate strands
Mixture contains DNA template, primers, nucleotides, and DNA polymerase.
Cooled and primers join the strands of existing DNA, then Taq polymerase synthesises new strands of DNA using the original strands as templates as many as 30-40 times, leading to over 1 billion exact copies of the original DNA segment

Question 13

Why is PCR important?

Answer 13

• Because significant amounts of a DNA sample are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification.

Question 14

How are plasmid vectors used in gene amplification?

Answer 14

A target gene is inserted in a circular plasmid, which is introduced into a bacteria
- Bacteria carrying the correct plasmid are selected using antibiotics and allowed to multiply to create many copies of the target gene
- Bacteria can be induced to express the gene and make protein e.g. the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics.

Question 15

What animals are used in receptor function and structure studies?

Answer 15

Ooctyes e.g. from frogs
Inject coding sequences into the oocytes and the animals grow and express the protein
E.g. Dr Philip Chen expressed different NMDAR subtypes and found they exhibit distinct biophysical properties

Question 16

What is protein crystallisation?

Answer 16

Cloning proteins can make sufficient protein to crystalise
- Once crystals have been created x-rays can be fired through them and the shapes of the proteins can be deduced via refraction patterns
- High resolution structural information allows for a greater understanding of drug-receptor interactions and is the basis of drug design

Question 17

What are DREADDs?

Answer 17

Designer receptors exclusively activated by designer drugs

Question 18

What are optogenetics?

Answer 18

Optogenetics is a biological technique to control the activity of neurons or other cell types with light. This is achieved by expression of light-sensitive ion channels, pumps or enzymes specifically in the target cells

Question 19

What can DREADDs be used for?

Answer 19

Can manipulate the DNA of a receptor so it binds a synthetic drug but not the original ligand
- Can insert this new channel in any tissue and render the channel to be activated by the synthetic drug
- This can be used to do a range of different things e.g. turn on excitable cells pharmacologically using synthetic ligands
- Different cellular pathways can also be switched on by a synthetic drug

Question 20

What are the tet-expression systems?

Answer 20

The Tet-on and Tet-off systems are two related inducible gene expression systems which allow researchers to control the expression of a gene of interest in a reversible manner by regulating the presence or absence of a specific inducer molecule, usually doxycycline or tetracylin .
When combined with CRISPR-Cas9,

Question 21

What are examples of different techniques for getting genes into cells?

Answer 21

Microinjection
Lipids
Electroporation
Biolistics
Viruses

Question 22

What are the pros and cons of microinjection for getting genes into cells?

Answer 22

Injecting DNA straight into the nucleas is efficient, it is a highly laborious procedure as only one cell at a time can be injected

Question 23

How are lipids used to deliver genetic materials?

Answer 23

DNA has a charge- this can be disguised by coating it in lipids to allow it to cross the cell membrane.
They are advantageous are they minimise side effects, and regulate how much material is delivered

Question 24

What are some examples of lipids being used to deliver genetic materials?

Answer 24

Lipid nanoparticals have shown success in the COVID-19 vaccines by moderna and Pfizer
Also shown to deliver siRNA for rare inherited diseases such as Onpattro, used for the treatment of polyneuropathy in people with hereditary transthyretin-mediated amyloidosis, a fatal rare disease that is estimated to affect 50,000 people worldwide

Question 25

What is electroporation?

Answer 25

The use of high-voltage electric shocks to introduce DNA into cells
Creates a high potential difference across membrane which creates holes in membranes- DNA can get in holes before they close up again.
Yields a high frequency of stable gene expression and requires fewer steps than other techniques

Question 26

What are biolistics?

Answer 26

• Biolistic transfection is a technique in which subcellular-sized particles coated with DNA are accelerated to high velocity to propel them into cells.
• Coating small particles of gold or titanium with DNA, high pulse of helium fires these out at high velocity and they can blast through the membrane to get to the nucleus ​

Question 27

What are thebdifferent viruses that are used to deliver genes?

Answer 27

Adeno-associated virus, adenovirus and lentivirus are all used and all have pros and cons

Question 28

What system can be used to knockout genes?

Answer 28

The Cre-lox system, derived from the P1 bacteriophage

Question 29

What is the Cre lox system?

Answer 29

The basic principle of the Cre-lox system involves the targeted insertion of LoxP sites into the genome on either side of a gene or genomic region of interest. When Cre recombinase is introduced into the organism, it catalyzes recombination between the LoxP sites, leading to various genetic modifications.
Need an organism containing loxP sites flanking the gene of interest and an organism containing Cre
Crossing the two strains will result in global deletion of the gene of interest

Question 30

How can you use the Cre-Lox system to selectively change genes?

Answer 30

Controlling Cre expression using a cell-specific promotor allows selective inactivation, activation, or mutation of genes of interest​

Question 31

What are the 3 types of gene arrangment that can occur due to the cre-lox system?

Answer 31

Inversion if the loxP sites are on the same DNA strand in opposite directions
Deletion if the sites face the same direction
Translocation if the sites are on seperate DNA molecules

Question 32

What is Cre recombinase and LoxP sites?

Answer 32

Cre is derived from the bacteriophage P1. It recognises and catalyses recombination at specific DNA sequences (LoxP sites) and acts like molecular scissors, cutting and rejoining DNA

LoxP sites are short DNA sequences that serve as recognition sites for Cre recombinase

Question 33

What system allows expression of genes in a spatial and temporal manner and how does it work?

Answer 33

The Cre/LoxP oestrogen receptor system
The Cre recobinase is fused with a modified ER that is only activated by tamoxifen.
Therefore, the Cre/LoxP system is only active when tamoxifen is present (e.g. spacial or timing)

Question 34

What system is being used increasinly to edit the genome?

Answer 34

The CRISPR-Cas9 system

Question 35

What is the CRISPR-cas9 system

Answer 35

CRISPR-Cas9 is a revolutionary genome editing technology that allows precise modification of DNA sequences in living organisms
Cas9 is an enzyme that acts as a molecular pair of scissors in the CRISPR system. It is guided by a small RNA molecule called guide RNA which is complementory to desired gene
Once the gRNA-Cas9 complex binds to its target DNA sequence, Cas9 cuts both strands of the DNA at a precise location determined by the sequence of the gRNA. Then mutations, corrections or new DNA can be inserted

Question 36

What therapeutic tools does CRISPR-cas9 have?

Answer 36

Gene editing: Modifying genes to study their function or correct disease-causing mutations.
Disease modeling: Creating animal or cell models of human diseases to study disease mechanisms and develop potential therapies.
Therapeutics: Developing gene therapies for treating genetic disorders, cancer, and other diseases.

Question 37

Can germline cells be edited in the UK?

Answer 37

Many of the proposed mechanisms for CRIPSR-cas9 involve editing the genomes of somatic cells however there is debate over their use in reproductive cells.
Gene editing in germline cells is currently illegal in the UK.

Question 38

What are CAR-T cells?

Answer 38

Chimeric antigen receptor T cells.
In this strategy, a patient’s own T cells are genetically engineered to express a synthetic receptor that binds a tumor antigen. CAR T cells are then expanded for clinical use and infused back into the patient’s body to attack and destroy chemotherapy-resistant cancer.

Question 39

Have CAR-T therapies been successful?

Answer 39

Since 2017, six CAR T-cell therapies have been approved by the FDA. All are approved for the treatment of blood cancers.
Despite the excitement around these therapies, they lead to long-term survival in fewer than half of the patients treated. They have also come under criticism for their cost, which, in the case of the most recently approved CAR T-cell therapy, is more than $450,000.

Question 40

Who was the Berlin patient?

Answer 40

Timothy Ray Brown- first person cured of HIV
Had acute myeloid leukemia and needed a stem cell transplantation as chemotherapy was not working. His doctor looked for a donor who had a mutation called CCR5 Delta 32 on the CD4 cells making them nearly immune to HIV. CCR5 is a protein on the surface of the CD4 cell that acts as doorway for the HIV virus to enter into the cell.
Got the stem cell transplant in 2007.

Question 41

How do the tet off system work?

Answer 41

There is a tetracycline controlled transactivator protein (tTA) which regulates expression of a target gene that is under transcriptional control of a tetracycline responsive promoter element (TRE) or
OFF= in the prescence of dox tTA cannot bind TRE and expression of the target gene remains inactive

Question 42

How does the tet on system work?

Answer 42

There is a a tetracycline controlled transactivator protein (tTA) which regulates expression of a target gene that is under transcriptional control of a reverse tetracycline-controlled transactivator, rtTA. The transcription of the TRE-regulated gene is stimulated by rtTA only in the presence of Dox.

Question 43

What is single cell sequencing and what is it used for?

Answer 43

Powerful molecular technique that allows the analysis of genetic characteristics of individual cells
Can study the genomic, transcriptomic, epigenomic, and proteomic profiles of individual cells.
Can be used to study genetic variants, mutations and genomic heterogeneity within a population of cells e.g. endothelial cells to study function

Question 44

What was a limitation of single cell sequencing until recently?

Answer 44

The readout of the sequencing does not preserve the spatial organisation of the cells in tissues

Question 45

When was the first cell single sequenced?

Answer 45

Single cell in 2009- by Tang et al
Since then, the technique has been developed so that 100,000s of cells can be sequenced at once.

Question 46

What is in situ barcoding?

Answer 46

In situ barcoding refers to a technique used in molecular biology and genomics to label and identify individual cells or molecules within their natural environment, typically within tissues or complex biological samples. The term “in situ” implies that the labeling and identification occur within the context of the original biological sample, preserving spatial information.

Question 47

What is the process for single cell sequencing?

Answer 47

Single cells, reverse transcription reagents, gel beads containing barcoded oligonucleotides are combined in a reacion vesicles called Gel beads in emulsion or GEMs
Within each reaction, reverse transcription of mRNA occurs with the barcoded oligonucleotides into solution
As a result, all cDNAs from a single cell will have the same barcode, allowing the sequencing reads to be mapped back to their original single cells of origin.
The prepared libraries are then subjected to high-throughput screening via platforms such as illumina or 10x genomics

Question 48

For single cell sequencing, what tissue can cell nuclei be taken from?

Answer 48

Can be taken from fresh or frozen tissue, although the readout is more specific in fresh

Question 49

What are single cell multi-omits?

Answer 49

Single cell multi-omics aim to provide a more holistic view of the cell by simultaneously masuring the genome and a more complete view of the interactions and combined functions
Can co-profile different aspects of the cell such as transcriptome and cell surface proteins to give a better indication of how the cell interacts and functions

Question 50

What is a drawback of single cell multi-omics data?

Answer 50

Lack of computational methods that can integrate across data modalities is a key issue in multimodal single-cell omics data analysis and greatly hinder biological discovery from such data.

Question 51

What is spacial transcriptomics?

Answer 51

Spatial transcriptomics is a groundbreaking molecular profiling method that allows scientists to measure all the gene activity in a tissue sample and map where the activity is occurring.
This can be particularly crucial for understanding the organisation of cells within tissues, studying tissue development, and identifying spatially distinct cell populations in heterogeneous samples.

Question 52

What is chain termination?

Answer 52

Standard PCR with the addition of modified nucleotides known as ddNTPs
- ddnTPs lack the 3’-OH group required for DNA bond formation therefore when DNA polymerase incorporates a ddNTP at random, the extension of the DNA ceases.
- The result is millions to billions of copies of the DNA sequence terminated at random lengths- these sizes are then separated by gel elecrophoresis.
- By reading the gel bands from smallest to largest we can determine the 5’ to 3’ sequence of the original DNA strand.

Question 53

How was chain termination sped up?

Answer 53

Chain termination or Sanger sequencing could read 1000 bases a day max- not useful when decoding the human genome.
Then the ddNTPs became fluorescently tagged and a computer could detect the resulting light emitted as each of the 4 ddNTPs had a different colour.
This helped speed up the process- a £200,000 machine could read 130000 bases a day. However this would have taken 63 years to decode the human genome.

Question 54

What is the next generation sequencing called?

Answer 54

Massively parallel sequence determination

Question 55

What are the different commercial methods used to do sequencing?

Answer 55

Roche, ABI SOLiD and Illumina

Question 56

What technique does Illumina use to sequence DNA?

Answer 56

Uses technology equivalent to parallel chain termination called bridge amplification

Question 57

What is oxford nanopore sequencing?

Answer 57

Generated recominant pores that a single strand of DNA can be pushed through via enzymes.
The bases are all different sizes and they effect what ions can get through the channels
When a base moves through the pore can measure the current to see what the base is

Question 58

What is an example of human emborys being modified with CRISPR/cas9?

Answer 58

In 2018 Dr He Jianhui targeted CCR5 in human embryos with the intention of making the children’s cells resistant to infection by HIV
- Caused an international outcry
- He altered embryos for for seven couples during fertility treatments, with one pregnancy resulting thus far. In each case, the father was infected with HIV; the mothers were HIV-negative
- However, transmission of HIV from the father’s sperm to the embryo, is a highly unlikely event​

Question 59

What is bridge amplification?

Answer 59

Sequencing technique by illuma
Fragments of DNA with adapters are denatured to form single strands
Strands complementary to the adapter sequence as tuck to the cell surface and therefore DNA gets stuck when they connect. They can also form bridges with complementary pairings of the adapters on the other end.
- Enzymes and dNTPs are added to form an immobilised double stranded DNA fragment. The second branch is then washed away and the process repeated.
- This causes many copies of the original DNA fragment to be created, and as the amplification process continues, clusters of identical DNA fragments are formed on the solid surface.
- Fluorescently labeled dNTPs are added one at a time, and as each nucleotide is incorporated into the growing DNA strand, its fluorescence is detected by a laser from a computer
- The dNTPs can be fluoresenctly labelled and a laser from a computer can identify the exact dNTP that was added based on the wavelength emitted by the fluorescent tag.
- From the billions of reads obtained by this process, software can determine the DNA sequence of the fragment by overlaying the sequencing data and comparing it to reference genomics.

Question 60

How much does Illumina’s bridge amplification technology cost?

Answer 60

Illumina’s bridge amplification technology was the first DNA sequencing technology to sequence the entire human genome for just $1000