week 3

Question 1

describe gel electrophoresis

Answer 1

• DNA is a negatively charged molecule at neutral pH
• therefore, in an electric field, will move towards the positive charge (anode)
• place a DNA preparation in a gel. Apply an electric field to pull the molecules through
• small pieces of DNA move faster than big pieces of DNA

Question 2

name and describe types of gel

Answer 2

agarose - polysaccharide from seaweed. suitable for coarse separation of quite large pieces of DNA (low resolution)
acrylamide - cross-linked polymer, suitable for very precise separation of DNA fragments that differ in length by as little as 1 base (high resolution). nasty neurotoxin!

Question 3

how to see DNA in an agarose gel

Answer 3

• use ethidium bromide (nasty mutagen) which acts as a fluorescent dye
• it is a flat molecule that intercalates between the stacked bases of DNA, causing the dye to display an increased florescence compared to free dye (in solution)
• UV radiation at 254 nm is absorbed by the DNA and transmitted to the bound dye
• the energy is re-emitted at 590 nm in the red-orange region of the spectrum
• is usually incorporated into the gel and running buffer
• the stain is visualised by irradiating with a UV light source (i.e using a transiluminator)
• the usual sensitivity of detection is better than 0.1 ug of DNA

Question 4

how to work out sizes of DNA fragments

Answer 4

• for agarose gel electrophoresis, we need a way to estimate the size of DNA fragments - use a collection of marker fragments of known size (e.g. HindIII digest of the lambda-phage genome)
• to work out the sizes of fragments, draw a graph of marker fragments by plotting log size versus distance migrated from well
• the plot is roughly linear over small size-ranges. then use the standard curve to estimate the size of your unknown DNA fragment. the comparison only works for linear DNA fragments

Question 5

3 states of plasmids

Answer 5

• supercoiled (the natural state)
• nicked (relaxed) circular
• linear
  they all migrate differently on agarose gels

Question 6

describe nucleic acid hybridisation

Answer 6

• an important and very useful property of single stranded DNA (and RNA) is it’s tendency to stick (or hybridise) to DNA molecules with a complementary sequence
• this property is exploited in many molecular biology techniques

Question 7

what is southern and northern blotting used for

Answer 7

• these techniques are a way to rapidly detect very small amounts of specific DNA or RNA sequence in gels
• detection is by hybridisation using a labeled (radioactive or fluorescent) probe - i.e a piece of DNA of known sequence that is easily detected.

Question 8

describe southern blotting

Answer 8

• hybridisation of a DNA probe to a DNA sample.
• DNA run on gel is usually whole genomic DNA digested with restriction enzymes.
• the DNA fragments on the gel are transferred to a membrane which is then incubated with a labelled probe that is complementary to the target DNA sequence.
• the probe is usually a known piece of DNA that has been radioactively/fluorescently labelled.
• the membrane is then washed to remove unbound probe.
• the temperature at which the probe is washed off of the membrane determines how similar the probe has to be to its target to hybridise.
• higher temp promotes more specific probe binding

Question 9

describe some applications of southern blotting

Answer 9

basic science:
- insertion of genes
- finding gene homologues in other organisms
- looking at genetic variation in populations
- finding clones in a DNA library - colony blots
biomedicine/diagnostics/forensics:
- detection of specific DNA sequences/polymorphisms. DNA fingerprinting
- fluorescence in situ hybridisation - e.g. uses in pre-natal screening

Question 10

describe northern blotting

Answer 10

-hybridisation of a DNA probe to an RNA sample
- a variation used for detection of specific RNA sequences
- make a preparation of mRNA molecules, run them on an agarose gel and blot them onto membranes
- then hybridise with a labeled DNA probe much as in southern blotting
- northern blotting tells you which genes are being transcribed in a particular organism/tissue under a specific set of circumstances
- used for monitoring levels of expression of specific genes

Question 11

why are expression arrays used

Answer 11

• northern blotting is very difficult
• extracting RNA is hard as it breaks up so easily
• the gels contain formamide and formaldehyde which are very toxic
• very lengthy process

Question 12

what are expression arrays

Answer 12

small glass/silicon chips that contain thousands of DNA probes. each probe corresponds to a specific gene. the chip is used to measure gene expression by detecting mRNA levels in a sample.

Question 13

describe the process of using expression arrays

Answer 13

• mRNA is isolated from samples and converted into cDNA.
• this is then fluorescently labelled
• the labelled cDNA is washed over the microarray and binds to complementary probes
• amount of fluorescence at each spot indicates how much mRNA (gene activity) was in the sample

Question 14

what are the uses of expression arrays and give an example

Answer 14

• cancer research - identifies which genes are over/under expressed in tumours
• drug development - see how a drug affects gene activity
• study how gene expression changes over time
  Say you’re comparing gene expression in normal vs. cancerous tissue:
• You extract mRNA from both.
• Label normal with green dye, cancer with red dye.
• Hybridize both to the same array.
• If a spot glows red, the gene is more active in cancer.
• If green, it’s more active in normal.
• If yellow, equal in both.

Question 15

describe the polymerase chain reaction

Answer 15

• PCR is a method for rapidly making millions to billions of copies of specific, short segments of DNA using very small amounts of starting template.
• the products of PCR can be used in a range of molecular biology applications, including DNA sequencing, identification of infectious agents, other forms of analysis and DNA manipulation

Question 16

what is template DNA

Answer 16

the source DNA to be copied/amplified

Question 17

what are primers

Answer 17

short pieces of single-stranded DNA that specify the region of the template to be copied/amplified

Question 18

what is taq polymerase

Answer 18

a heat-stable DNA polymerase used to synthesize new DNA strands by adding nucleotides to the 3’ ends of primers, essentially replicating the DNA

Question 19

what is denaturation

Answer 19

the separation of strands in a double-stranded DNA molecule by heating (95 degrees)

Question 20

what is annealing

Answer 20

the hybridisation of primers to template DNA by cooling to an appropriate temperature (65 degrees)

Question 21

what is extension

Answer 21

the synthesis of double-stranded DNA from single-stranded DNA with an annealed primer. done by taq polymerase at (72 degrees)

Question 22

what are dNTPs

Answer 22

a mixture of dATP, dGTP, dCTP, dTTP, the ‘building blocks’ of DNA

Question 23

what is a thermal cycler

Answer 23

a heater block that rapidly heats and cools sample tubes. controls the temps needed for pcr

Question 24

which direction is DNA replication and what does it require

Answer 24

• always in the 5’ to 3’ direction as one end has 5’ phosphate while other end has 3’ OH group. DNA polymerase can only add nucleotides to a free -OH group so synthesises from 5’ to 3’. (adding to 3’ end)

requires single-stranded template so polymerase can read it and add complementary bases. needs double-stranded section usually created by a primer to latch onto. and free 3’ end OH group for DNA polymerase to start elongating the strand.

DNA polymerase uses dNTPs to add nucleotides to growing DNA strand. it reads the template strand and adds complementary bases. moves along the template in the 3’ to 5’ direction but synthesises the new strand in the 5’ to 3’ direction.

Question 25

what are PCR reaction ingredients

Answer 25

- template DNA - dNTPs - 2 primers - one forward and one reverse primer making the top and bottom strand - thermostable/taq polymerase - buffer solution, providing a suitable chemical environment for polymerase activity - MgCl2 to help the enzyme function

Question 26

in theory vs in practice for PCR

Answer 26

In theory, the amount of target DNA doubles with each round but in practice, PCR is never this efficient. However, 1 single PCR reaction of 35 cycles will usually produce enough amplified DNA for most forms of genetic analysis

Question 27

what happens to the long bits

Answer 27

- over successive rounds, more and more of the newly synthesised strands will be of length equal to the distance between the two primers - this is because over successive rounds more of the single-stranded templates will themselves be the products of previous rounds of amplification

Question 28

problems with PCR

Answer 28

contamination - PCR is extraordinarily sensitive, especially when one performs many cycles. Just a few contaminating molecules can give misleading results. Inappropriate priming - PCR primers can initiate DNA replication even if they don't match a piece of template DNA perfectly (especially if they do match the template DNA at the 3' end of the primer). This can produce additional DNA products or sometimes, completely the wrong products Taq errors - Taq polymerase-mediated DNA synthesis is error-prone (~0.5% errors). i.e Taq will sometimes put the wrong base in the DNA chain it is synthesising Optimisation - factors such as Mg concentration, primer concentration and annealing temperature often need to be optimised before a PCR can work efficiently Difficult sequences - some DNA sequences are difficult to amplify, especially GC rich sequences

Question 29

other points about PCR

Answer 29

- the polymerase obviously plays a vital role. The discovery of thermostable polymerases that are not degraded during the heating stage of the reaction was crucial - the primers can be modified to introduce specific changes into the amplified DNA sequence.

Question 30

other considerations in PCR primer design

Answer 30

- annealing temperature matching - length and GC content - primer dimers - hairpins - repeat sequences / unique sequences - GC clamps

Question 31

what is annealing temperature matching

Answer 31

- during the annealing stage of PCR, if the temperature goes below the annealing temperature of the primer, then it will anneal to non-target locations - it is therefore important for the 2 primers to have similar annealing temperatures

Question 32

describe length and GC content

Answer 32

- GC pairs stick more strongly than AT pairs. the more G and C a primer has, the higher the annealing temperature - the longer a primer is, the higher the annealing temperature - so we can control the annealing temperature of the primer by making it longer or shorter and by choosing primers with higher or lower GC content

Question 33

describe primer dimers

Answer 33

primer dimers are unintended short DNA fragments that form when primers hybridise to themselves or one another at their 3’ ends

Question 34

describe hairpins

Answer 34

-hairpins are primers that have complementary sections within themselves primers that form hairpin structures can be less efficient - hairpin structures with a 5' overhang can lead to further extension from the 3' end creating a nonsense primer

Question 35

describe repeat sequences/unique sequences

Answer 35

- should avoid primers that anneal to multiple locations throughout the genome and many different PCR products will be produced - better to target unique sequences

Question 36

describe GC clamps

Answer 36

many people argue that it is important to have at least one C or G in the last 5 bases of a primer to increase binding stability at the 3’ end and prevent non-specific binding