DNA Replication and PCR

Question 1

DNA molecules can be…

Answer 1

… separated and annealed.

Question 2

Why can dna strands be easily separated?

Answer 2

because hydrogen bonds are weak

Question 3

the strands can be easily
separated because hydrogen
bonds are weak - and then be
made to…

Answer 3

…reanneal.

Question 4

the strands can be easily
separated because hydrogen
bonds are weak - and then be
made to reanneal

this is important in gene
expression because…

Answer 4

… its how genes are transcribed.

Question 5

the double helix can be ‘melted’ forming what?

Answer 5

2 single stranded DNA polymers.

Question 6

How are genes transcribed in eukaryotes?

Answer 6

as single units.

Question 7

In eukaryotes genes are transcribed as single units however in bacteria genes involved in the same
pathways are often arranged linearly as an operon

When the operon is transcribed all of the genes are transcribed in one continuous mRNA known as a
polycistronic mRNA

What does this increase the efficiency of?

Answer 7

This increases the efficiency of gene regulation

– for instance if the bacterial cell needs to make biotin it makes sense to transcribe all of the genes involved in this process together

–means there can be a simple genetic switch to turn the process on.

Question 8

DNA is polymerised by…

Answer 8

… a complex of proteins at the replication fork.

Question 9

What is the function of dna polymerase?

Answer 9

catalyses the addition of bases to the 3’ end

Question 10

Whats the function of Topoisomerase?

Answer 10

unwinds the helix to prevent torsion

Question 11

Funcition of helicase?

Answer 11

melts the double stranded molecule

Question 12

Function of DNA primase?

Answer 12

makes short RNA primers (gives a free 3’ end)

Question 13

Whats the function of single stranded binding proteins?

Answer 13

prevents premature annealing

Question 14

Whats the function of DNA ligase?

Answer 14

Joins okazaki fragments on the lagging strand.

Question 15

DNA strands run…

Answer 15

…antiparallel

Question 16

Each strand of the double helix actually is antiparallel to its…

Answer 16

… complementary strand.

Question 17

Each strand of the double helix actually is antiparallel to its complementary strand.

Why does this come about?

Answer 17

Because DNA is polymerised only in one direction (5’ to 3’). Nucleotides are added at the 3’ end. This is where DNA polymerase joins, and generate from 5’ to 3’.

Question 18

The 5 and 3 here refer to the…

Answer 18

carbon atoms on the deoxyribose sugar that joins with phosphate groups.

Question 19

DNA polymerase needs small ‘primers’ – short sequences of RNA (made by RNA polymerase) to…

Answer 19

…start it off.

Question 20

In order to synthesise new strands of DNA several things are needed:

Answer 20

1) A complementary strand of DNA (template DNA)
2) An excess of short oligonucleotide primers to provide a free 3’ end
3) Molecules of the 4 bases: dATP, dCTP, dGTP, and dTTP
4) A dna polymerase enzyme (Taq polymerase - thermostable)
5) Magnesium ions (Mg2+) - a co-factor for all DNA polymerases.
6) Buffer to maintain pH to correct value for DNA polymerase.

Question 21

How can we amplify dna?

Answer 21

By using the previously described elements and cycling between denaturing, annealing and extension temperatures we can amplify DNA from a few molecules to many molecules.

Question 22

Describe dna amplification?

Answer 22

Starting with very few copies after each round of amplification there is a theoretical doubling of the number of DNA molecules

Question 23

After only 30 cycles of denaturing, annealing and extension 1 original
molecule will result in…

Answer 23

… 1 billion DNA molecules.

Question 24

Describe step 1 of PCR?

Answer 24

1) Denaturation

By heating the DNA to 95°C we can denature the double strand because the weak hydrogen bonds are broken, so the two strands are separated.

Question 25

Describe step 2 of PCR?

Answer 25

2) Annealing

If we have in excess lots of primer molecules, then those primers will anneal to their complementary sequence.

Question 26

Describe step 3 of PCR

Answer 26

3) Extension

If we now add in a polymerase and the nucleotides dATP, dCTP, dGTP, dTTP then the complementary strand will
polymerise.

Question 27

What needs to be done in order to understand how a multicellular organism works?

Answer 27

we need to identify which genes are turned on at
which tissues at certain times.

Question 28

Understanding how a multicellular organism actually
works we need to identify which genes are turned on at
which tissues at certain times.
In this example we may want to examine gene expression
from mouse embryos at different developmental stages –
What will this give us an insight into…

Answer 28

which genes are important at different stages of development (genes that show
temporal regulation)

Question 29

How do we look at whether a gene has been turned on or off in an organism?

Answer 29

1) by examining RNA levels
2) by examining protein levels.

Question 30

When examining RNA levels or when examining protein levels, what are looking at?

Answer 30

the gene product to see whether a gene has been turned on or off.

Question 31

To study gene expression, what technique can we use?

Answer 31

RT-PCR (reverse transcription-PCR)

Question 32

RT-PCR (reverse transcription-PCR) involves…

Answer 32

… the formation of complementary DNA (cDNA) to mRNA in the cell, remember mRNA has a poly-A
tail.

Question 33

Why does RT-PCR (reverse transcription-PCR) work?

Answer 33

the technique works because:
1 - single stranded DNA will base pair to single stranded RNA
2 – the enzyme reverse transcriptase can polymerase DNA using an RNA
template

Question 34

a complementary DNA strand (cDNA) is produced after the addition of…

Answer 34

… a poly-T primer

Question 35

mRNA has a…

Answer 35

… poly-A-tail

Question 36

after first strand synthesis of reverse transcription, we are left with…

Answer 36

… a ssDNA molecule (RNA is digested)

Question 37

Once we are left with our ssDNA, if we now add two primers and Taq polymerase we can…

Answer 37

… do a PCR amplification

Question 38

how is cDNA formed?

Answer 38

cDNA is formed from mRNA, reverse transcriptase, and a poly-T-primer

Question 39

What’s the purpose of a “no DNA template” or “No Template Control” lane in PCR?

Answer 39

A No Template Control (NTC) lane in PCR experiments ensures that there is no contamination in the reagents or in the environment. The NTC lane consists of all the PCR components but with no DNA template. If there is any amplification in this lane, there has been contamination. This could lead to false-positive results. Having this lane ensures the PCR results are accurate.

Question 40

Purpose of a positive control in PCR?

Answer 40

Ensures that the PCR is capable of giving a positive result. This is done using a sample which is known to give a certain result. If the positive control gives its expected outcome, researchers know the PCR is working correctly. If it does not produce this expected result, they know the setup is not functioning correctly and there is a problem.

Question 41

What is the purpose of genomic dna lane in pcr?

Answer 41

Used as a reference or control to make sure pcr setup is working correctly. Contains genomic dna where the target dna sequence is from. Researchers can compare the results from the genomic dna lane and the lane of the target DNA sequence to ensure the pcr is working correctly and efficiently.

Question 42

purpose of doing a pcr of actin?

Answer 42

The purpose of performing a PCR of actin, specifically beta-actin, is to use it as a loading control. Beta-actin is a housekeeping gene that is consistently expressed at stable levels across different cell types and conditions. This makes it an ideal reference to ensure that the amount of sample loaded in each lane is consistent. By comparing the expression of beta-actin to the target gene, researchers can normalize the data and account for any variations in sample loading or experimental conditions

Question 43

What is one major problem of RT-PCR?

Answer 43

one major problem of RT-PCR is that it is only semi-quantitative.

Question 44

in the PCR reaction, once many
molecules of amplified product have been made they begin to…

Answer 44

…compete with primers during the annealing step – so less product is made

Question 45

How does RT-PCR work?

Answer 45

the mechanics of PCR combined with reverse transcription – various
enzymes, buffer, dNTPs, primers – 3 step PCR process after RT, then gel electrophoresis.

Question 46

Why does RT-PCR work?

Answer 46

because DNA forms double
stranded molecules by
complementary base pairing – Taq polymerises new strands but needs a template etc.

Question 47

Why would we use RT-PCR?

Answer 47

in order to study where or when a gene is being expressed – gene regulation

Question 48

What does RT-PCR tell us?

Answer 48

qualitative or semi-quantitative
patterns of gene expression

Question 49

What does RT-PCR NOT tell us?

Answer 49

real quantitative data of
expression – also whether the
gene gets translated to protein – or how active the protein is.

Question 50

What other techniques other than RT-PCR would give us more information?

Answer 50

qPCR, northern blotting,
transcriptomics, studies of the
protein itself

Question 51

What is the solution to RT-PCR’s major problem?

Answer 51

qPCR

Question 52

Rules of designing primers?

Answer 52

1. obviously the sequence has to be complementary
2. length of primer – usually around 20-25 bases (the longer it is the higher the
   annealing temperature and the more specific it is)
3. at least 50% of the bases should be G or C (because they have more hydrogen
   bonds – they increase the annealing temperature)
4. there should not be repeats within the bases
5. the primers shouldn’t be complementary to each other
6. if possible the 3’ (last) base should be a G or C
7. the melting temperatures (Tm)of the two primers should be similar

Question 53

To express the protein, you need to design primers to the …

Answer 53

… ‘Open reading frame’ (contains no stop codons).