Outcome 4

Question 1

DNA sequencing refers to

Answer 1

the sequencing of nucleotides in a DNA molecule

Question 2

What is another name for Sanger sequencing

Answer 2

Chain termination

Question 3

List the components required for Sanger sequencing

Answer 3

Template DNA with a known short sequence that flanks the 3’end. Primer, heat stable DNA Polymerase, dNTPs, ddNTPs

Question 4

What are the differences between dNTPs and ddNTPS?

Answer 4

ddNTPs lack a 3’ OH group. This means that chain elongation is terminated as soon as a ddNTP is incorporated into the chain. This is because DNA polymerse catlayses the formation of phophodiester bonds between the 5’ phosphate group of one nucleotide and the 3’OH group of the next.

Question 5

How are ddNTPs modified for Sanger Sequencing

Answer 5

They are modified to be covalently bonded to fluorescent tags. So they can be identified.

Question 6

Why is only one primer used for Sanger Sequencing

Answer 6

Unlike PCR, Sanger Sequencing does not seek to amplify target DNA. Focusing on one strand, Sanger Sequencing it aims to synthesise a complementary strand of every possible length.

Question 7

Describe the first step in Sanger Sequencing?

Answer 7

Template dsDNA, a primer, heat stable DNA poymerase, dNTPs and ddNTPs are mixed together. The each of the 4 types of ddNTP are covalently bonded to floursecent dyes and these 4 flourescent dyes emit light at different wavelengths. The ratio concentration of dNTPs in the mixture is much greater than the concentration of ddNTPs.

The mixture is heated to 95C, the H bonds between the bases of dsDNA are broken, it denatures into ssDNA.

Question 8

Describe the second step in Sanger Sequencing

Answer 8

The mixture is cooled to 45-60C. Primers, which are present in excess, bind via complementary base pairing to their recognition sites on the template ssDNA. These recognition sites are located on the 3’ flank of the template ssDNA.

Question 9

Describe the third step in Sanger Sequencing

Answer 9

The mixture is heated to 72C. Heat stable DNA polymerase uses the primer as a starting point and begins catalysing the formation of phosphodiester bonds between the free 3’OH group of the primer and the 5’ phosphate group of a dNTP or ddNTP that is complementary to the correspoing base on the template ssDNA. The length of the extension depends on when a ddNTP is incorporated into the chain, at which point nucleotide chain elongation will cease.

Given enough time, reagents, and a suitable ratio of dNTPs to ddNTPs, at least one DNA strand of every possible length will be produced with a
fluorescent ddNTP at the end.

Question 10

Describe the analysis step of Sanger Sequencing

Answer 10

Each strand is seperated on size using capillary gel electrophoresis. The smallest fragment will reach the capillary end first, a flourescent detecting laser shooting through the gel will excite the flourescent tag on the ddNTP of the fragment and the wavelength of the light emitted by the terminal ddNTP can be measured. The second shortest fragment will then reach the capillary end and the same process will occur. Once all fragments have reached the capillary end, the sequence of the full length of DNA can be determined. The sequence of the detected fluorescence is then converted computationally into an electropherogram.

Question 11

Why is capillary gel electrophoresis used instead of agarose gel electrophoresis

Answer 11

Capillary gel is 3 times faster, uses less material and acheives better seperation.

Question 12

What is an electropherogram

Answer 12

A graph showing the intensity of flourensence emmision over time. in Sanger Sequencing it is used to show the nucleotide sequence.

Question 13

How is Sanger Sequencing used to detect mutations?

Answer 13

Sanger Sequencing is used to detect single nucleotide variants. It relies on comparision between a patients electropherogram and the electropherogram of a DNA sample that does not contain the mutation. Differences in the electropherograms are analysed for phenotypic effect.

Question 14

What are two uses of Sanger Sequencing

Answer 14

1. To determine a nucleotide sequence

2. To detect single nucleotide variant mutations

Question 15

What are the features of a microarray

Answer 15

It is a biochip containing thousands of highly ordered spots arranged into rows and columns. Each spot contains multiple copies of ssDNA called probe DNA, that could for example represent a gene or a portion of a gene or a previously known mutation. The location and composition of each spot is recorded in a database.

Question 16

How is a microarray used to determine whether a patient has a mutation for a specific disease?

Answer 16

A DNA sample from the patient and a control DNA sample which does not contain a mutation of the gene of interest are collected and undergo the same process seperately from one another.
The DNA is denatured into ssDNA.
The ssDNA is cut into smaller fragments and subsequentlly labelled with fluourescent dye.
Different label used for patient and control.
Both sets of labelled DNA are mixed and inserted into the chip. The chip is incubated to allow for hybridisation to occur.
Excess DNA is washed off the chip.
DNA hybridisation can be identified by a microarray scanner which measures flouresence.
If patient has no mutation, both control and patient DNA will bind to the sequences on the chip that represent the normal sequence. If flourescence matching the patients DNA label is detected on the spots which contain mutation fragments then they have a mutation.6

Question 17

What is gene expression profiling

Answer 17

Using a microarray to study gene expression levels in cells and /or tissue.

Question 18

In gene expression profiling what is used as an indicator of gene expression

Answer 18

mRNA

Question 19

How is mRNA isolated?

Answer 19

The cell is lysated and affinity chromotography with oligo dT beads is used

Question 20

Describe the steps in gene expression profiling

Answer 20

Cells or tissue of interest are lysated and mRNA is isolated from the lysate using affinity chromotography with oligo dT beads. The isolated mRNA is used to produce cDNA using RT-PCR.
The cDNA is amplified and labelled with flourescent dye.
cDNA can then be inserted into the microarray and incubated for hybridisation. The spots on the microarray contain DNA probes which correspond to specific mRNAs. The microarray scanner detects flourescence emission at specific spots.

Question 21

What is gene mapping

Answer 21

Methods used to identify the location of a gene within a chromosome and the distances between genes.

Question 22

What is a linked gene

Answer 22

When genes are close to one another on the same chromosome they are called linked.

Question 23

What is linkage

Answer 23

When alleles located on the same chromosome are inherited together

Question 24

What is recombination

Answer 24

Recombination is when portions of DNA in a homologous chromosome cross over at random, break and recombine during the first phase of meiosis. Thus producing new allele combinations.

Question 25

What determines the frequency of crossover between 2 genes during recombination

Answer 25

The distance between the 2 genes - further apart = more likely to crossover.

Question 26

What is linkage mapping

Answer 26

A form of gene mapping where the recombination frequency of genes is used to produce a linkage map

Question 27

Summarise how 2 genes on a fruit fly can have their recombination frequency calculated.

Answer 27

A fly that is homozygous for both dominant alleles is mated with a fly that is homozygous for both recessive alleles. The F1 offspring are double heterozygous.
The F1 offspring is crossed with a fly that is homozygous for both recessive alleles. This is a test cross - guarantees that the alleles provided by F1 offspring determine phenotype.
Recombination frequency is calculated by adding up the number of non-parental phenotypes in the F2 offspring. This number is divided by the total number of F2 offspring and then x100.

Question 28

Appart from linkage mapping what is another method of gene mapping

Answer 28

Physical mapping (sequencing)

Question 29

What are two uses of linkage mapping

Answer 29

Used to understand the genetic basis of disease - can be used to identify well known gene linked to disease causing allele.

Used in genomic sequencing - draft genomes must be assembled into final genome. Linkage maps used as framework for genome assembly

Question 30

What is a genetic marker

Answer 30

A gene or other DNA sequence with a known location on a chromosome, that can act as a landmark for identification purposes.

Question 31

What are microsatellites

Answer 31

Tracts of DNA that are 1-10 nucleotids long. These nucleotide tracts repeat 5-50 times in a row. (Tandem repeats). Because microsatellites occur at thousands of places within the genome and the physical location of many microsatellites is known. They are important markers

Question 32

What are 3 applications of microsatellite markers

Answer 32

Paternity tests, forensics and linkage mapping

Question 33

Why are microsatellite markers used in paternity tests

Answer 33

Microsatellites have a much higher mutation rate than codind sequences and show high variation between individuals. A child is likely to have similar microsattellites to their mother and father but distinctly different microsatellites to non-relatives.

Question 34

How are microsatellites used for paternity tests

Answer 34

DNA samples from the mother, child and proposed father are taken and multiple microsatellites can be seperated and amplified via PCR. They are analysed via agarose gel electrophoresis and compared.

Question 35

How are microsatellites used for forensics

Answer 35

DNA profiles can be generated by amplifying and seperating a series of microsatellites from the crime scence. DNA from suspects can be processed in the same way and compared to help identify who was present at the crime scene.

Question 36

What were the aims of the Human Genome Project

Answer 36

1. Determine an accurate sequence for a composite human genome
2. Determine the number of genes in the human genome
3. Sequence genomes of other medically relevant organisms
4. Develop analystical tools
5. Store info on public databases
6. Anticpate and prepare for legal, ethical implications arising from availability of genome information availability

Question 37

What is BAC-BAC sequencing

Answer 37

A modified version of Sanger sequencing used for the HGP.

Question 38

Describe the 1 step of BAC-BAC sequencing

Answer 38

A physical map of each chromosome was generated -
Many copies of the chromosome were randomly cut into fragments of 150kbp in length. The fragments were inserted into Bacterial Artifical Chromosomes for amplification. The amplified fragments were analysed for the presence of markers and also resctriction sites via restriction enzymes. The restriction maps were aligned to create a physical map of the chromosome.

Question 39

Describe the 2 nd step of BAC-BAc sequencing

Answer 39

Each BAC fragment is further fragmented into 1,500 pieces and each piece is placed in a M13 vector. As M13 vectors is already known, the fragment sequence is easily identified.
The fragment sequences are then assembled into orded using a computer programme that identifies regions of overlap. Once all 150,000bp fragments are fully sequenced they can be correctly ordered using a physical map.

Question 40

Describe whole genome shotgun sequencing

Answer 40

Multiple copies of the genome are broken into 2,000 bp pieces by squeezing the DNA through a pressurised syringe. A fresh sample of the genome is broken into 10,000 bp pieces using a similar approach.
Each fragment is inserted into a plasmid.
THe resulting libraries are sequenced.
The millions of sequenced fragments are assembled into longer fragments called contigs.
The contigs are assembled into a continous stretch of DNA corresponding to each chromosome.