Characterisation of genes and gene products at a molecular level

Question 1

DNA cloning definition

Answer 1

the process of making multiple identical copies of a particular piece of DNA.

Question 2

Process of DNA cloning

Answer 2

1. Restriction endonuclease cuts DNA at specific recognition sequence.
2. Restriction endonuclease cuts complementary DNA at plasmid.
3. DNA ligase joins the two fragments in the presence of calcium chloride which makes bacterial wall more permeable to the DNA fragment.
4. DNA then multiplied rapidly in PCR, thus amplifying the DNA.

Question 3

Special features of the recognition sequence

Answer 3

Often 6 bases long and produces a palindromic sequence.

Question 4

What is a palindromic sequence?

Answer 4

Read the same 5’-3’ as 3’-5’

Question 5

Features of cloning vectors

Answer 5

• Contain an antibiotic resistance gene for selection of the plasmids that contain the new gene.
• contains an origin of replication for the replication of the plasmid
• multiple cloning site
• phage promoters which enable in vitro transcription of the recombination DNA fragment

Question 6

Explain DNA electrophoresis

Answer 6

electric field is established across a gel, causing the DNA molecules to move a certain distance dependent on their size

Question 7

Why would DNA be different lengths?

Answer 7

Contain many recognition sites for different restriction endonucleases which results in the DNA being cleaved at different base pair lengths.

Question 8

Stages of DNA electrophoresis

Answer 8

1. Electric field established across the gel
2. DNA will move to anode, as it is negatively charged due to phosphate groups
3. Polymers forming the gels have a porous matrix, which acts as a sieve allowing shorter DNA to move more rapidly than larger ones.
4. DNA chains are then visualised using different methods

Question 9

What gel is used for electrophoresis?

Answer 9

polyacrylamide and agrose

Question 10

Two different methods of visualising DNA

Answer 10

1. Stain with ethidium bromide, a fluorescent compound that binds to DNA. Forms red-orange fluorescent bands under UV.
2. Autoradiography- Radioisotope 32P is incorporated into DNA before electrophoresis, then placed on a radiographic film after migration is complete.

Question 11

Hybridisation of nucleic acids process

Answer 11

1. Double stranded DNA molecules denatured by heat

2. Complementary sequences re-hybridise at lower temperatures

Question 12

3 processes based on DNA hybridisation

Answer 12

southern blotting, northern blotting, fluorescent in situ hybridisation (FISH)

Question 13

Stages of Southern Blot

Answer 13

1. DNA sample cleaved by a restriction endonuclease
2. Placed onto a polyacrylamide and agrose gel and separated by electrophoresis
3. Gel is immersed in sodium hydroxide to denature the DNA
4. DNA transferred from gel onto another support, such as a sheet of nitrocellulose. This is done by adding layers of absorbent paper soaked in salt solution into a cuvette. Gel block containing DNA is placed over the paper block and the nitro-cellulose sheet is arranged at the top.
5. Dry blotting paper is added to the top, causing the solution from the lower stack to rise, moving the DNA in the gel to the nitrocellulose.
6. Nitrocellulose, now a replica of the DNA, is heated in an oven to fix and immobilise the DNA onto this substrate

Question 14

What occurs next and what is the function of southern blotting?

Answer 14

Single stranded DNA probes containing attached radioactive labels of 32P then hybridise with the complementary DNA on the nitrocellulose.

Sheet washed to remove unhybridised DNA.

Photographic film placed onto the nitrocellulose to shoe bands of DNA where the probe has attached,

Question 15

What is northern blotting?

Answer 15

Same as southern blotting however finding specific RNA sequences

Question 16

What is western blotting?

Answer 16

Same as southern and northern blotting instead proteins used

Question 17

What is FISH?

Answer 17

Molecular cytogenetic technique that uses fluorescent probes that bind to specific complementary sequences on the DNA.

Question 18

FISH function

Answer 18

Localises specific DNA sequences on chromosomes, so can be used in genetic counselling, personalising medicine and species identification.

Question 19

What can be used to correct the mutated/non functional sequence?

Answer 19

CRISPR - clustered regulatory interspaced short palindromic repeats

Question 20

What is CRISPR-Cas9?

Answer 20

Consists of a repetition of short sequences of DNA separated by spacer sequences that, along with the Cas9 endonuclease, correspond to an adaptive immune system that protects the bacteria from the invasion of virus or plasmids,

Question 21

CRISPR-Cas9 DNA technology use

Answer 21

Can be used to add bases or disrupt DNA sequences to alter specific genes with greater accuracy, simplicity and lower cost

Question 22

Two ways in which Cas9 nuclease breaks the double strand of DNA?

Answer 22

Homology Directed Repair- gene knock in

and Non-Homologous End Joining- gene knock out

Question 23

Explain Homology Directed Repair

Answer 23

1. RNA sequence in CRISPR complementary to the DNA. Cas 9 utilises the sequence to bond with the base pairs on the host DNA.
2. Cas 9 causes a double stranded break.
3. DNA template then invades the gap , using the DNA’s synthesis machinery to generate a complementary code.
4. A segment of DNA from the template strand may be inserted into original break.
5. Template separates leaving the original break with a new section of code.

Question 24

Explain non-homologous end joining

Answer 24

1. DNA cleaved by Cas 9
2. Either new matching bases added or non-matching bases excised
3. joins the ends using ligase
4. gene no longer functions

Question 25

3 enzyme based techniques

Answer 25

Polymerase chain reaction, reverse transcription polymerase chain reaction and DNA sequencing

Question 26

PCR function

Answer 26

Unlimited amplification of a DNA fragment of interest from a very small amount of a starting template material

Question 27

PCR stages

Answer 27

1. DNA to be amplified sequenced from oligonucleotide fragments 2. Added to a thermocycler where temperature is 95 degrees, causing the DNA chains to separate 3. Temperature lowered to 50 degrees, enabling the annealing of primers. 4. Temperature raised to 72 degrees, enabling taq polymerase to catalyse the condensation of adjacent nucleotides. DNA ligase then joins the Okazaki fragments. 5. Cycle is repeated

Question 28

RT-PCR stages

Answer 28

1. Reverse transcriptase enzyme catalyses the formation of a DNA strand from an RNA template. 2. A TTTTTTTTTT oligonucleotide primer can be used to transcribe the entire mRNA into DNA, called cDNA 3. amplify cDNA using PCR

Question 29

Function of RT-PCR

Answer 29

Detects and estimates the amount and quality of mRNA

Question 30

Process where nucleotide sequence of a particular DNA fragment is determined

Answer 30

DNA sequencing

Question 31

Who discovered the principle of DNA sequencing? + what did he do?

Answer 31

Sanger- determined the nucleotide sequence of insulin

Question 32

Sanger DNA sequencing requirements

Answer 32

1. Requires single stranded DNA template, DNA primer, DNA polymerase, normal deoxynucleotidetriphosphates dNTPs and modified di-deoxynucleotidetriphosphates, ddNTPs.

Question 33

Sanger DNA sequencing requirements

Answer 33

1. Requires single stranded DNA template, DNA primer, DNA polymerase, normal deoxynucleotidetriphosphates dNTPs and modified di-deoxynucleotidetriphosphates, ddNTPs. A lot more dNTPs are present than ddNTPs in order to completely transcribe the DNA ddNTPs are tagged using fluorescent dye, each a different colour

Question 34

Sanger DNA sequencing explained

Answer 34

Using the templates, the complementary DNA strand is being synthesised, however ddNTPs prevent the chain from being further extended, thus producing different lengths of complementary chain. DNA then denatured whilst heating to release the different length strands. Then undergo gel electrophoresis and placed onto a radioactive film. The relative positions of the terminating ddNTPs determine the sequence of DNA, from lightest (start) to heaviest (end)

Question 35

Different uses of antibodies in protein detection

Answer 35

Western blotting, Elisa, Imaging and Immunoprecipitation

Question 36

Western Blotting process

Answer 36

1. Proteins denatured 2. Proteins within the sample are separated using gel electrophoresis, depending upon their molecular mass, electric charge 3. Moved from gel to nitrocellulose membrane through the process of electroblotting. 4. Membrane is blocked in non fat dry milk or bovine serum. Protein in the solution bind to the membrane in all spaces where the target proteins have not attached. 5. antibody is then added to specific target protein and then incubated, washed to remove buffer and unbound antibodies, 6. secondary antibody added, linked to probe 7. probe deleted using fluorescent detection or radioactive detection.

Question 37

Western Blot function

Answer 37

Used to detect specific proteins in a sample

Question 38

What does ELISA stand for?

Answer 38

Enzyme linked immumosorbent assay

Question 39

ELISA steps

Answer 39

1. Antigen added to plate, given time to adhere 2. milk added to bind everywhere where antigen is not 3. primary antibody with conjugated enzyme added, binds to antigen 4. plate washed 5. substrate to enzyme added, creating a colour change 6. the higher the concentration of primary antibody present, the stronger the colour change 7. colour changed measured by spectrometer

Question 40

ELISA function

Answer 40

Diagnostic tool in medicine

Question 41

What is ELISA used to screen?

Answer 41

HIV, HIV antigens added, if HIV antibodies present in serum then imdividcl may have HIV.

Question 42

How are antibodies used in imaging?

Answer 42

Antibodies for a specific protein fluorescently linked | Antibody recognises receptor and binds, glows, can then be detected

Question 43

Immunoprecipitation definition

Answer 43

The technique of precipitating a protein antigen out of a solution using an antibody that specifically binds to that particular protein

Question 44

Explain direct immunoprecipitation

Answer 44

1. antibodies immobilised onto a specific solid phase, such as a string of breads, such as microscopic agarose 2. The beads are then added to the protein mixture where they bind to their targets and are captured

Question 45

high throughput defined

Answer 45

automation of experiments such that large scale is feasible