Introduction To Molecular Techniques

Question 1

Role of restriction enzymes

Answer 1

• they recognise speechifying sequences of DNA known as palindromic sequences.
• they cut DNA at these specific sites

Question 2

What is a palindromic sequence

Answer 2

• a sequence that is the same when read from the 3’ to 5’ and 5’ to 3’ direction

Question 3

What are the 4 requirements for gel electrophoresis

Answer 3

1) gel : ( often agorose gel) which is a matrix that allows separation of DNA fragments
2. Buffer : allows maintenance of the charge on the DNA samples across the gel
3. Power supply : generates charge difference across gel
4. Stain/detection : to identify presence of separated DNA.

Question 4

Where on the electrophoresis gel plate is DNA placed ?

Answer 4

At the negative electrode because it is negatively charged so would need to move towards the positive electrode

Question 5

Why do we use gel electrophoresis?

Answer 5

1) to investigate the size of DNA fragments
2. To investigate mutations
3. To investigate DNA variations
4. To clone DNA

Question 6

What are the5 basic steps of cloning a gene

Answer 6

1) isolate relevant gene of interest following digestion with restriction enzymes
2. Insert gene of interest into plasmid vector - same restriction enzyme is used to cut the vector.
3. Ligation : DNA ligase forms RECOMBINANT DNA by forming phosphodiester linkages between the nucleotides of vector and gene.
3. Introduce recombinant dna molecule into suitable host cells
4. Identify and isolate the clone containing the DNA of interest

Question 7

Why do we clone human genes ?

Answer 7

1) to make useful proteins eg insulin
2. To find out what genes do
3. Genetic screening
4. gene therapy

Question 8

Outline the steps in polymerase chain reaction

Answer 8

1) denaturalising: the strands must first be separated. DNA is heated to 95 degrees - this breaks the hydrogen bonds and causes DNA to unwind.
2. ANNEALING : temp is lowered to around 50 degrees to allows PCR primers to bind to the exposed strand of DNA.
3. Extension : the process of adding new complementary nucleotides using Taq polymerase ( a thermostable polymerase which can tolerate the high temperatures). The temperature is raised to 75 degrees. This forms duplicate double helix.

Question 9

Wha what is the function of PCR?

Answer 9

• Amplification of target DNA
• investigate single base mutations
• to investigate small deletions or insertions
• investigate variations

Question 10

On what basis are proteins separated?

Answer 10

• size
• shape
• charge
• they will move towards the anode or cathode if placed in an electric field

Question 11

What are the requirements for protein gel electrophoresis

Answer 11

1. Gel : a matrix that allows separation of the protein sample
2. Buffer : maintains charge on the protein samples
3. Power supply : generates a charge across the gel
4. Stain / detection : to identify the presence of separated proteins

Question 12

What is the role of serum protein electrophoresis?

Answer 12

• measures specific proteins in the blood to help identify some diseases.
• serum protein electrophoresis uses an electrical field to separate the proteins in the blood serum into groups of similar size , shape and charge.

Question 13

Why SDS-PAGE separates proteins on the basis of size ?

Answer 13

SDS separates proteins primarily by mass because the detergent ( SDS) denatures and binds to proteins to make them uniformly negatively charged.

• thus when a carried is applied , all SDS bound proteins in a sample will migrate through the gel towards the positively charged electrode

Question 14

Why do we use enzyme assays ?

Answer 14

• important tools for measuring cellular activity of enzyme kinetics.
• provides us useful information of the mechanism of enzyme catalysis and interactions of enzymes with substrates , drugs etc.

Question 15

How will you measure the product in continuous enzyme assay ?

Answer 15

• chemoluminescence

- spectrophotometery

Question 16

How would you measure the product during discontinuous enzyme assay ?

Answer 16

• radioactivity

- chromatography

Question 17

How can antibodies be used in immunoassays to detect the presence of a protein?

Answer 17

• it is used to detect and quantify the amount of protein.
  1. An antigen must be immobilised first to a solid surface,
  2. Antibody that is linked to an enzyme then binds to the antigen.
  3. Incubated with a substrate to produce a measurable product. Enzyme converts substrate into coloured product. The rate of colour formation is proportional to the amount of specific antibody
  4. The more antibodies that bind = more protein there is.

Question 18

How can antibodies be used in western blotting to detect the presence of proteins ?

Answer 18

1. protein gel is transferred on a nylon membrane
2. Add antibody which binds to specific protein
3. Use another antibody ( eg fluroscent one) to detect first antibody .

Question 19

Where do restriction enzymes come from ?

Answer 19

Bacteria

Question 20

What type of primers do we need during the annealing process of PCR?

Answer 20

• we need primers that go in the opposite direction , not the same direction,
• they need to actually span the gene on the DNA strand

Question 21

Outline the steps of southern blotting - a technique of DNA hybridisations

Answer 21

1) digest DNA with a restriction enzyme
2. Separate DNA with gel electrophoresis
3. The gel is soaked in an alkaline solution which denatures the DNA into ssDNA molecules ( single stranded).
4. The separated fragments of ssDNA now need to be transferred to a solid support : usually nylon or nitrocellulose filter is used.
5. Once the DNA has been transferred onto the nylon filter the gel can be discarded.
6. The filter is now placed in a solution containing labelled DNA probe ( Radioactive or fluorescent labelled). These probes will bind to ssDNA on the filter with a complementary sequence.
7. After binding the probe , we wash the filter to remove any unbound probe.
8. Detect binding using photographic film or UV light for fluorescent detection methods.

Question 22

Why do we use southern blotting ?

Answer 22

1. To investigate variation
2. Investigate mutations
3. Investigate gene expansions ( triplet repeats)
4. Investigate gene structure ( large deletions or duplications)
5. Allows us to detect very small amounts of DNA that may not be visible by staining of DNA in a gel.

Question 23

What are important characteristic of DNA probes in southern blotting ?

Answer 23

• the probes do not have to have 100% similarity to the target sequence - it will just bind less tightly
  2. Probes do not have to completely align with the sequence they are being used to detect
  3. Probes do not affect the position of the target sequence on a gel

Question 24

What is the Sanger chain termination method

Answer 24

• allows us to work out the nucleotide sequence of a piece of DNA

Question 25

What is the structural difference between deoxynucelotide triphopshate and dideoxynucelotide triphosphate ?

Answer 25

Deoxynucleotide triphosphate has a typical 3’ hydroxyl group.

• whereas with dideoxynucleotide triphosphate there is no -OH group on the 3’ carbon , only a H.
• this is important because it prevents further elongation as no phosphodiester bonds will be formed with a subsequent dNTP.

Question 26

What are the ingredients for Sanger sequencing .

Answer 26

• DNA polymerase
• primer
• the four dna nucleotides ( dATP , dTTP, dCTP, dGTP)
• the template DNA to be sequenced
• dideoxynucleotides ( ddATP, ddTTP, ddCTP, ddGTP) each labelled with a different coloured dye

Question 27

Outline the steps required for Sanger sequencing

Answer 27

1) 4 separate test tubes each with the unique ddNTP and a labelled primer to initiate DNA synthesis
2. PCR must occur first
3. The ddNTP terminate the reaction and you get DNA fragments of different lengths
4. The ends of the fragments will be labelled with dyes that indicate their final nucleotide
5. After the reaction is done the fragments are run through a long , thin tube containing a gel matrix in a process called capillary gel electrophoresis
6. We are then able to read off the sequence from the bottom of the gel to work out the nucleotide sequence.

Question 28

What is the role of 2-mercaptoethanol (2-ME)

Answer 28

• it breaks dislufied bridges between collagen molecules , so molecular weight would decrease and protein would move further down the electrophoresis gel plate