DNA

Question 1

What ‘materials’ are required in order to conduct sanger sequencing?

Answer 1

Template DNA you want to sequence
A short DNA primer
DNA polymerase
Nucleotides including dideoxynucleotides

Question 2

Describe the method sanger sequencing up until the production of collections of DNA strands of different lengths

Answer 2

The mixture of template DNA is heated to denature the DNA, then the temperature is lowered which allows the DNA primer to bind to its complementary sequence on the template DNA. The temperature is raised again allowing DNA polymerase to bind and start creating a new strand of DNA. The enzyme makes no distinction between standard and dideoxy-nucleotides and each time a deodioxynuclotide is incorporporated, DNA synthesis stops. This reaction occurs billions of times as there are billions of DNA molecules present in the test tube and in each reaction, incorporation of a dideoxynucleotide happens at a different random point This results in the collection of many DNA strands of differing lengths

Question 3

Describe the method sanger sequencing from the points at which collections of DNA strands of different lengths have been produced to the ‘reading’ of the DNA sequence

Answer 3

The sequencing reaction is transferred to a lane of polyacrylamide gel which is placed into a DNA sequencer for electrophoresis and analysis. The fragments migrate according to size and each is detected as it passes a laser beam at the bottom of the gel. Each type of dideoxynucleotide (A,C,T,G) emits light of a characteristic wavelength and is recorded as a coloured band on a simulated gel image. The computer interprets the row data. and outputs an electropherogram with coloured peaks representing each letter in the sequence. The simulated gel image is read from bottom to top, starting with the smallest fragment. In this way, the sequence of DNA can be ‘read’

Question 4

What is meant by the term ‘next generation sequencing’?

Answer 4

Next generation sequencing uses new technologies to provide rapid sequencing of complex genomes very rapidly. Importantly, it can sequence multiple genes simultaneously.

Question 5

Describe the method of Illumina next generation sequencing up until the point of production of multiple nests of DNA on the flow cell

Answer 5

Randomly fragmented pieces of DNA are produced and tagged with an adapter at either end of the fragment. The adapters act to ID each DNA fragment and also act as a primer. The DNA is then added to a flow cell which also contains pre-bound adapters that are complementary to one of the adapters bound to the DNA. This allows the formation of bridges. to create a very. short sequence of ds-DNA that can be extended by adding nucleotides and DNA polymerase. This creates ds-DNA which is then denatured to produce two complementary strands of ss-DNA. This process is then repeated indefinitely, so that over time a ‘nest’ of identical strands is formed at that point on the flow cell. This process occurs for different DNA strands a billions of sites across the flow cell, to create billions of ‘nests’ each containing a different DNA sequence

Question 6

Describe the method of Illumina next generation sequencing from the point of production of multiple nests of DNA on the flow cell to the point of sequencing

Answer 6

The nests of DNA are sequenced. One base at a time is added and these. bases are fluorescently tagged. A laser then scans across the slide, so that each type of base (A, T, C, G) flashes a different colour and this is recorded by a computer. The slide is then washed, another base is added and the. slide is gained again. This is repeated many times so that the DNA sequence is slowly built up.

Question 7

Fidelity (the ability of the sequences to be accurate) is not as good in illuminati next generation sequencing than in sanger sequencing. How does the computer attempt to overcome this?

Answer 7

The computer reports an average of the DNAA sequence produced from all the individual strands of DNA within one ‘nest’/cell within the flow cell. The computer will also report how precise the ‘average’ report is

Question 8

The sequences produced from next generation sequencing are very small, how is this then mapped to create an overall picture of the genome?

Answer 8

The small fragments of DNA that are sequenced are aligned to give the ‘overall picture’ of the genome. This alignment is done by looking for areas of overlap. The computer program will identify these overlaps to build-up as long a sequence as possible.

Question 9

What is nano pore sequencing and what are its advantages and disadvantages?

Answer 9

This is a portable DNA sequencing technique that can be used in the field. It can give much longer sequences of DNA than other methods but is not as accurate and can be more expensive.

Question 10

Compare and contrast sanger, Illuminaten and nano pore sequencing in terms of accuracy, number of genes sequences, length of DNA sequenced, cost and location of use?

Answer 10

Sanger - very accurate, single reaction good for looking at a defined region, read max 1000 basepairs, cheap, lab based

Illumina - some errors, massively parallel (can look at whole genome), reads short sequences ~150 basepairs, cheap and lab based

Nanopore - error prone, massively parallel (can look at whole genome), reads 10,000 base pairs on average, more expensive, portable

Question 11

Describe the method of a polymerase chain reaction

Answer 11

PCR starts by denaturing DNA to separate it into single strands. Next, the temperature is lowered allowing the left and right primers to anneal to their complementary sequences. The primers are designed to ‘bracket’ the DNA which is to. be sequenced. The temperature is raised to allow tax polymerase to bind at each primary site and extend (synthesise) a new DNA strand. The DNA is then denatured again to separate it into single strands, the temperature is lower,d primers bind, the temperature is raised, tax polymerase binds and a new DNA strand is synthesised. This process Is repeated for many cycle and with each cycle the number of copies of the DNA of the target length/sequence increases exponentially.

Question 12

What is the use of looking at the cycle time at which the halfway mark on the PCR graph is reached?

Answer 12

This will give an idea of how much DNA was present originally in a sample. The more DNA - the shorter the time to reach the halfway mark

Question 13

How can PCR be used to detect single gene anomalies?

Answer 13

If there is exact knowledge of the gene target then PCR can be used to amplify the DNA fragment of interest and then gel electrophoresis can be used to identify missing exons.

Question 14

Give an example of a condition in which PCR be used to detect single gene anomalies

Answer 14

Duchenne’s muscular dystrophy

Question 15

A large part of DNA does not code for genes and within this section of the genome there are some highly repeated elements. What might be included here?

Answer 15

Long and short interspersed nuclear elements
Remnants of retroviruses
DNA transposon fossils

Question 16

Describe how chromosome crossover can occur. during meiosis?

Answer 16

During the process of sexual reproduction, the homologous chromosomes align during the division of meiosis to produce gametes. During this process there is shuffling of chromosomes termed crossover where regions of

Question 17

Describe how chromosome crossover can help identify genes associated with / that cause disease?

Answer 17

The likelihood of crossover between genes is dependent on how physically close they are to one another on the chromosome: crossover is more likely when there is greater physical distance. So, if we wanted to study a particular disease gene, whose location is unknown, we can look at markers whose location is known. If the disease gene and marker are rarely co-inherited, this suggests that they exist far apart whereas if they are often co-inherited, this suggests that they. exist close together.

Question 18

Describe how microsatellites can help identify genes associated with / that cause disease?

Answer 18

Microsatellites are repeat elements within DNA that can act as a marker. Microsatellites have now been mapped across the genome and so if we then study large families, we can identify which microsatellites are associated with the disease and thus, where the disease gene is likely to exist. PCR can be used to amplify different microsatellites and measure their length.

Question 19

Give examples of conditions in which microsatellites were used to help identify disease genes

Answer 19

Duchenne’s muscular dystrophy

Huntington’s disease

Question 20

Describe how SNPs are use in whole genome association studies

Answer 20

Whole genome sequencing has allowed the identification of SNPs across the genome. If you know where SNPs are found in the genome, you can design a flow cell on which to conduct next generation sequencing. You only need to add one nucleotide and sequence this to produce an entire SNP map for an individual. If this was done for many individuals (some with a disease and some without), you can then detect which SNP variants are associated with the disease. This can help to identify. genes (by looking at which genes exist near the SNP) that are associated with the. disease. Thus, this can generate hypotheses on possible disease-causing genes.

Question 21

What is population stratification (in terms of DNA)?

Answer 21

Systemic differences in allele frequencies between sub-populations.

Question 22

How can we protect against population stratification in gene association studies?

Answer 22

Controls for ethnic make-up for cases
Repeat the study in different populations
Statistical methods sullied to allow a correction factor for population structure

Question 23

What is epigenetics?

Answer 23

Heritable changes that are not caused by changes in DNA sequences. This suggests that there are differences in programming of DNA in order to give rise to genetically identical cells which are all differentiated for different functions

Question 24

Give an example of a method which gives rise to the phenomenon of epigenetics?

Answer 24

DNA methylation which can turn transcription on or off

Question 25

In eggs and sperm epigenetic programming is removed from cells. Why is this important?

Answer 25

So that the fertilised egg can develop into any. type of cell. Then as the embryo develops, epigenetic changes are accumulated and differentiated cells form.

Question 26

Give an example of a condition caused by abnormalities in epigenetic programming?

Answer 26

Beckwith Widermann syndrome. Here there is an abnormality in CTCF which binds to and inactivates enhancers, causing overexpression of a genes -IGF2.