26-10-21 - Introduction to Molecular Biology 2

Question 1

What are the 3 different modes of how organisms may replicate their DNA?

What method was DNA found to replicate in?

What experiment proved this?

Answer 1

• Semi-conservative model - where a strand of DNA serves as a template to make a new, complementary strand
• Conservative model – one molecule consists of both original strands and new model consists of 2 new strands
• Dispersive model – 2 molecules that are hybrids of parental and daughter DNA – patchwork of original and new DNA
• DNA was found to be replicated using the semi-conservative model
• This was proved by the 1983 Meselson-Stahl experiment

Question 2

Describe the Meselson-Stahl experiment from 1983

Answer 2

• Initially E.coli began growing in a nutrient broth containing a heavy isotope of Nitrogen (N15)
• The bacteria used this N15 to synthesize new biological molecules, including DNA
• After many generations, the nitrogenous bases of the bacteria’s DNA were all labelled N15
• The bacteria were then transferred to a regular nitrogen environment (N14) and allowed to grow several generations
• They began synthesising DNA using N14
• Meselson and Stahl knew when E.Coli divided, so could take samples after each generation and purify the DNA
• The measured the density of the DNA (and indirectly its N14 AND N15 content) using density gradient centrifugation
• This method separates DNA molecules into bands by spinning them at high speeds
• This allows the differences between N14 AND N15 to be detected.
• Densito-metric scans could be produced to determine the method by which DNA replicates, which was found to be the semi-conservative model.

Question 3

How is speed achieved when it comes to DNA replication?

Answer 3

• DNA replication starts at many places simultaneously, with the replication fork moving in both directions

Question 4

Why are strands of DNA unwound during replication?

How does this happen?

What direction does DNA replicate in and why?

How do the leading and lagging strands replicate?

What leads to the replication forks being asymmetrical?

Answer 4

• Due to the nature of the replication fork, the DNA needs to be unwound during replication
• This is done by DNA Helicase, and sometimes topoisomerases are used to decrease DNA contortion
• DNA only replicates in the 5’ to 3’ direction due to DNA polymerase
• The leading strand is replicated continuously towards the replication fork
• The lagging strand is replicated discontinuously away from the replication fork in Okazaki fragments
• Since DNA replication occurs in both directions in both strands, there will be leading and lagging strand templates on the replication fork at both sides.
• This leads to the replication forks being asymmetrical

Question 5

What needs to be done to allow for enough room for Okazaki fragments to be made?

How is this stabilised? What is generated after every 150 bases are exposed and why?

What happens when DNA polymerase reaches the RNA primer of the previous Okazaki fragment?

Answer 5

• In order to provide enough room for Okazaki fragments to be generated, single strands have to be exposed, which is done by DNA helicase.
• This is stabilised temporarily by DNA binding protein
• After every 150 bases of single strand template being exposed, DNA primase generates an RNA primer, so a new Okazaki fragment can be made
• RNA primers are short templates that provide the free 3’ end that is needed for DNA polymerase to add new bases to form an Okazaki fragment
• RNA primers are also required for synthesis of the leading strand, but only 1 initial RNA primer is needed, as opposed to many for the lagging strand
• When DNA polymerase reaches the RNA primer of the previous Okazaki fragment, the RNA primer is degraded and replaced by DNA
• The gap between the Okazaki fragments is the joined together by ligase.

Question 6

What problem can arise with the lagging strand at the telomeres?

What is the role of telomerase?

What cells is telomerase most/least active in?

What happens if these mechanisms are not in place?

What condition would this cause?

Answer 6

• At the telomeres, there may not be the 150 bases left on the single template strand that are required to stimulate DNA primase to produce another RNA primer in order to produce the lagging strand
• This problem is resolved by Telomerase
• Telomerase brings with it a guide RNA template, which can add some information to the template of the lagging strand in the form of TTAGGG sequences repeated.
• DNA polymerase can now come and generate an Okazaki fragment
• Telomerase is most active in stem cells and proliferating cells, and least active in somatic cells (any cells other than reproductive cells)
• Without these mechanisms to replicate DNA found in the telomeres, this DNA would become unstable and degrade, leading to the shortening of the chromosomes
• This would lead to Werner’s syndrome, which results in premature aging

Question 7

How often do polymerases make mistakes?

What can happen if these mistakes are not corrected?

What are 2 methods where errors can be removed?

What can errors in these systems cause?

Answer 7

• Polymerases make mistakes (e.g incorporating wrong base – G with T) as often as once every 10,000 polymerizations (monomer addition) events
• This can lead to mutation fi they are not corrected before the next round of replication
• Errors can be removed during synthesis
• DNA polymerase has a polymerization site that adds bases, then an editing site that proofreads the bases added
• If the wrong base is added, the editing site will crop out the incorrect base, and insert the correct base.
• Mutations can also be repair post-replication via DNA mismatch repair
• Certain families possess defects of this DNA mismatch repair system, which pre-disposes them to disease due to an accumulation of mutations

Question 8

What is polymerase chain reaction (PCR) used for?

How does the process work?

Answer 8

• PCR is used to generate lots of copies of genetic material (DNA) from little starting material (amplify genetic material)
• DNA strands are heating in order to break the hydrogen bonds and separate the strands
• RNA primers of known sequence can be synthesised and added to the mixture
• DNA likes to exist in a double stranded conformation, so the individual DNA strands will find and hydrogen bond to their matching RNA primers
• DNA polymerase and monomers complementary to the DNA strand can then be added to the mixture, which results in the synthesis of new DNA.

Question 9

How is PCR used in the presence of infectious agents?

What are 5 advantages to this technique?

Answer 9

• A sample can be taken from an infectious person
• The genetic information from the infection is separated and purified, then amplified using primers
• Gel electrophoresis can then be used to determine if someone is infected, with a control using blood from a non-infected person also included.
• Advantages of this technique:
• Fast compared to culturing
• Can give specific identity of strain
• Can tell us severity of infection, as it gives a quantitative product in a predictable way (e.g how many PCR cycles for accumulation of infectious agent to occur)
• Can be used to see if treatment is working.

Question 10

How is PCR used to identify inheritance patterns?

When is this useful?

Answer 10

• We can see inheritance of particular chromosomes using PCR
• This is useful if we know a particular disease is associated with changes on a specific chromosome
• Repeated sequences on paternal and maternal chromosomes can be amplified and compared to their offspring’s repeated sequences via gel electrophoresis to see if the child has inherited any particular conditions

Question 11

How can PCR be used to check for changes in copy number of chromosomes?

Answer 11

• Quantitative fluorescent PCR can be used to identify changes in copy numbers of chromosomes

Question 12

How is DNA sequenced?

Answer 12

• Deoxyribonucleoside triphosphate (nucleotide) can be modified to form dideoxyribonucleoside triphosphate (modified nucleotide) by removal of the hydroxy group at carbon 3.
• When added to the chain, this prevents dideoxy from linking to another phosphate and forming a 5’ – 3’ phosphodiester bond, meaning the chain can not be synthesised any further from this point.
• This is used by DNA polymerase to stop synthesis
• A test tube can be filled with single DNA strands with labelled primers on them, deoxy nucleotides, and few dideoxy nucleotides
• DNA synthesis will begin to occur, and will be stopped at different points for each strand due to the dideoxy nucleotides, creating different lengths of strands
• These strands can then be put through gel electrophoresis, which allows the base sequence from 5’ to 3’ to be identified

Question 13

How is automated sequencing useful when looking for mutations?

What does this identification of mutation aid in?

Answer 13

• Automated sequencing is useful when looking for disease in particular chromosomes
• Heterozygous genes can easily be seen, where there is a different gene on each chromosome in a pair, which is caused by a mutation
• This can help confirm diagnosis and develop personalised medicine to target the specific mutative products

Question 14

What direction does the growing strand grow in?

Why is this?

What determines which bases will be added?

How are bases added to the chain? What is this reaction catalysed by?

Answer 14

• The growing strand is synthesised in a 5’ to 3’ direction
• This is because DNA polymerase only works in the 5’ to 3’ direction
• The template strand dictates what base is coming next (A with T and C with G)
• There are monomers known as dNTPs (deoxynucleotide tri-phosphate (nucleotides)), which consist of a deoxyribose sugar, 3 phosphates, and a base
• The hydroxide group at carbon 5 of the dNTPs will attach the carbon 3 of the chain to form the 5’ – 3’ phosphodiester link
• The formation of phosphodiester bonds is catalysed by DNA polymerase