S3: Introduction to Molecular Biological Techniques

Question 1

What is the building block of DNA?

Answer 1

It is a nucleotide. It consists of a pentose sugar (deoxyribose), a phosphate group and a nitrogenous base.

Question 2

Describe structure of a strand of DNA

Answer 2

• A single strand has a sugar-phosphate backbone.
• Phosphodiester bonds join the nucleotides. The phosphates link the 5’ carbon of one sugar to the 3’ carbon of the next.
• We read DNA in the 5’-3’ direction.
• 1’ is the carbon that the base is attached to and we continue counting clockwise.

Question 3

What is the process restriction?

Answer 3

Bacteriophages are viruses that infect bacteria so bacteria evolved to try and get rid of these viruses. It does this through the use of enzymes that recognise viral DNA and digests it thus killing the bacteriophages.

Question 4

Why are only type II restriction enzymes useful to us?

Answer 4

Type II restriction enzymes are sequence specific so they are useful in biological techniques.

Question 5

What are the two things restriction enzymes do?

Answer 5

1. They recognise specific DNA sequences

2. They then cut phosphodiester bonds in that sequence of adjacent to it

Question 6

Describe how recombinant DNA can be formed using the restriction enzyme EcoR I as an example

Answer 6

EcoR I will recognise a specific base sequence in the DNA. Once it has recognised it, it will cut it out by catalysing the hydrolysis of phosphodiester bonds. Depending on where the enzyme cuts, it may leave a few bases that are unpaired (single strands for a while - sticky ends). These bases are completely exposed so if they come into contact with their complementary sequence they can reanneal. The enzyme DNA ligase joins the annealed fragments together by forming new phosphodiester bonds.
DNA molecules from different sources can be ligated together to produce recombinant DNA. If two different DNA have been cut with EcoRI which produces sticky ends that are complementary they will become recombinant DNA.

Question 7

What are overhangs?

Answer 7

Some restriction enzymes (nucleases) produce overhangs known as sticky ends. The overhangs are useful as they can be used to reform double strands.

Question 8

What is restriction mapping?

Answer 8

They are maps of restriction sites within a molecules and is a way of working out how a plasmid or other unknown piece of DNA fits together. A plasmid restriction map is built using gel electrophoresis to seperate the DNA strands by size.

Question 9

How do we analyse the DNA fragments produced by restriction enzymes?

Answer 9

Gel Electrophoresis. The samples are put in wells and current it is added. Negatively charged DNA migrates towards the positive end (anode). They move in proportion to their size. We also put known sizes of DNA in so they can act as a control/standard in which other pieces of unknown DNA is compared to.

Question 10

What are the 2 stages of DNA amplification by cloning?

Answer 10

• Cloning in molecular biology is creating a DNA molecule of interest plus replication signals so that it can grow/replicate inside the organism.
• The DNA is then inserted into a organism to grow large numbers and to isolate the DNA. Organisms that are small and fast growing are ideal for this e.g. bacteria, yeast or viruses.
• The plasmids are able to replicate independently of the bacterial DNA.

Question 11

Describe plasmids

Answer 11

Plasmids are mostly circular nucleic acid molecules.
They occur naturally in bacteria and yeast and often contain beneficial genes e.g. antibiotic resistance that are beneficial to the host.

Question 12

Describe how the structure of plasmids make them beneficial for cloning

Answer 12

• Plasmids have a replication of origin that will allow the plasmid to be replicated when it is inserted into a host (i.e. so cloning can take place).
• The OriR (origin of replication) signal requires host’s enzyme for replication
• There are useful genes e.g. gene encoding antibiotic resistance such as ampicillin. This allows selectivity in the cloning process so we are left with only host cells that contain the plasmids we want.
• Finally there is the cloning region, this is where we can insert our foreign DNA.

Question 13

Describe how a recombinant plasmid is made using BamHI restriction enzyme as an example

Answer 13

• One site is cut with the BamHI restriction enzyme (it is important that this site does not appear again in the plasmid or multiple sites will be cut). This occurs in the cloning region of the plasmid.
• Cutting the plasmid with BamHI will produce sticky ends and then we cut the foreign piece of DNA with BamHI producing complementary sticky ends.
• The isolated gene will anneal with the plasmid and DNA ligase will catalyse this.
• The recombinant plasmid will then contain the gene that we want to clone.

Question 14

Describe how recombinant plasmid is transported/transformed into a host (as it is unable to replicate on its own)

Answer 14

Typically a harmless lab strain of E.coli is used as a host. This is because it grows rapidly in simple medium and it is easy to introduce the plasmid DNA at high efficiency and then extract the plasmid DNA after we’ve isolated. The plasmid is introduced into the host bacterium (called bacterial transformation) by making small pores in the cell wall. We do this by washing the bacteria with CaCl2 or by electroporation (zapping with electricity).

Question 15

Breifly describe the whole cloning process

Answer 15

1. Isolate the DNA
2. Isolate the region of interest using restriction enzymes
3. Ligate into plasmid
4. Transform into E.coli
5. Grow transformed bacteria
   6a. Induce protein expression in bacteria and then purify product
   6b. Purify plasmid DNA from bacteria and recover inserted foreign DNA using restriction enzyme

Question 16

What is PCR?

Answer 16

PCR stands for polymerase chain reaction.

• It is a good method to increase DNA on a massive level
• It is exponential amplification of a DNA fragment of which you need to know the ends of the sequence.

Question 17

List components (reagents) needed for PCR

Answer 17

• Template - The DNA we want to amplify
• Primers which are short pieces of ssDNA that need to be complementary to strand
• Polymerase which needs to be thermodynamically stable e.g. Taq
• Nucleotides
• Buffer to maintain pH
• MgCl2 for polymerase activity

Question 18

What is PCR thermal cycling?

What three processes result from this?

Answer 18

This is where the mixture of reagents are passed through three different temperatures over and over again. It goes up to 94 degrees and then down to 60 degrees and then up to 72. This is one cycle which then repeats.
This results in three processes: Denaturation, Annealing and Extension

Question 19

Describe PCR cycle

Answer 19

Starting point: Template DNA which contains DNA region we  want to amplify.
Step 1 (Denaturation): We heat and seperate the DNA strands to 95 degrees.
Step 2 (Annealing): The reaction is cooled to 5-60 degrees so the primers can preferentially anneal to our template DNA (to the start of the sequence we want to clone). We make sure there is excess primers so it allows them to bind rather than DNA re-annealing.
Step 3 (Extension): Heat up to 72 degrees so Taq DNA polymerase sees the primers and extends the strand from primers by adding complementary bases in the 5' to 3' direction.
The result is fragments of the DNA region of interest being amplified (2 identical copies). This repeats until reagents run out. The growth is exponential (DNA keeps doubling with each cycle - 2n).

Question 20

Why is PCR useful?

Answer 20

• It is useful because there is no need for the laborious cloning process to amplify DNA
• PCR requires very little starting material
• PCR is highly specific
• PCR can introduce restriction sites at ends which can later be cloned
• PCR can be scaled up to handle large numbers of samples

Question 21

Describe how restriction digestion can be used to see where bases have changed in DNA (use test for sickle cell as example)

Answer 21

For sickle cell anaemia we test for mutation in the beta globin gene (HBB).
1. DNA sample obtained.
2. PCR used to amplify region of beta globin gene.
3. Restriction digest the gene with the enzyme that would be sensitive to a mutation (in this case Ddel).
4. Gel electrophoresis of the products.
For someone with normal beta globing gene, the restriction site for Dde1 is shown. For a sickle cell gene, the sequence is different so it will not be recognised and thus not cut by the restriction enzyme.
- The allele with a mutation will appear heavier on gel electrophoresis as it is not cut
- A sickle cell sufferer will be homozyhous for the mutated gene and neither alleles wil have restriction site.