Using Gene Sequencing

Question 1

What is the genome?

Answer 1

The total of all the genetic material in an organism

Question 2

Where is DNA found in prokaryotes?

Answer 2

In the cytoplasm. Both in the main chromosome and the plasmids

Question 3

Where is DNA found in Eukaryotes?

Answer 3

In the nucleus of the cell and in mitochondria and in green plant cells DNA is also in the chloroplasts

Question 4

What are exons?

Answer 4

The coding regions of DNA (the genes)

Question 5

What are introns?

Answer 5

The large non-coding regions of DNA that are removed before messenger RNA is translated into proteins

Question 6

What is DNA/ gene sequencing?

Answer 6

The analysis of the individual base sequence along a DNA strand or an individual gene

Question 7

What do you do in gene sequencing?

Answer 7

Analyse individual strands of DNA or individual genes, giving us a pattern of bases that codes for a particular protein in the cell

Question 8

What do you do in DNA profiling?

Answer 8

Analyse the patterns in the non-coding areas of DNA (introns) and use them to identify individuals

Question 9

What is the Polymerase chain reaction (PCR) used to do?

Answer 9

Amplify a sample of DNA to make more genetically identical copies of it very rapidly

Question 10

Wwhat does it mean when we say that DNA has been amplified?

Answer 10

It is replicated repeatedly using the polymerase chain reaction to produce a much bigger sample

Question 11

When would you need to use PCR to amplify DNA?

Answer 11

In a crime investigation there may only be a minute DNA sample available and you need at least 1ug of DNA for DNA profiling (equivalent of DNA from 10 000 human cells)

Question 12

Why did scientists initially find it hard to amplify tiny amounts of genetic material?

Answer 12

The DNA sample needed to be heated to around 90-95°c to seperate the strands. However this destroys the DNA polymerase from most organisms

Question 13

Kary Mullis solved the problem of developing PCR. How did he do this?

Answer 13

He used enzymes from a bacterium that lives in hot springs. Because the enzymes in this bacterium has evolved to survive in the extreme conditions he hypothesised that they would be robust enough to cope with the high temperatures needed to seperate the DNA strands

Question 14

What are the stages of PCR?

Answer 14

1) the following are added to the thermocycler:
- the DNA fragment to be copied
- Taq DNA polymerase
- nucleotides
- primers
2) the thermocycler heats the mixture up to 95°c. This causes the hydrogen bonds between the bases to break causing the 2 strands to seperate
3) the mixture is cooled to 55°c. This causes the primers to join with their complementary base pairs at the end of each DNA strand. DNA polymerase attaches to the double strands.
4) - the temperature is increased to 72°c this is the optimum temperature for DNA polymerase. It adds complementary each DNA strand. Once the 2 strands are completed the process is repeated. The number of DNA strands increase exponentionally. Each cycle takes around 2 mins to complete

Question 15

What are primers?

Answer 15

Small sequences of DNA that must join to the beginning of seperated DNA strands before copying can begin

Question 16

What are the basic principles seen in all the different methods of DNA sequencing?

Answer 16

• the DNA strands are chopped into smaller pieces
• the double strands are seperated to give single strands
• PCR is involved in replicating the DNA fragments to produce large quantities of material for analysis
• labelled terminator bases are added to the single strands of DNA. A terminator base is a modified version of one of the A,T,C,G nucleotide bases. When a terminator base is incorporated into a DNA molecule the chain is halted as no more bases can be added. A T terminator stops DNA synthesis where a T base would be added ect. Each type of terminator base has a fluorescent tag in a specific colour
• the coloured tags enable the sequence of bases to be read very rapidly by an automated system

Question 17

What is massively parallel sequencing?

Answer 17

A very rapid method of sequencing millions of DNA fragments at the same time

Question 18

What are current sequencing technologies heavily dependent on?

Answer 18

State of the art computer technology. The raw data is fed into computer systems that can reassemble the genomes. They compare the fragments and find the areas of overlap between them

Question 19

How can scientists use DNA sequencing to determine the protein produced from any gene and what realisation has this led to?

Answer 19

Because of the universal code we can recognise the start and stop codons in a gene. Analysing the base pairs enables us to work out which amino acids will be joined together to form a protein as a result of information contained in the DNA.

This has led to the realisation that genes will code for more than one protein. And some genes will code for factors that affect the expression of other genes changing the protein product

Question 20

How can DNA sequencing help us to gain a better understanding of diseases that result from a mutation in a single gene?

Answer 20

DNA sequencing makes it possible to identify a faulty gene, see which bases have changed and understand hoe the changes in the DNA affect the protein produced. This then allows us to begin to work out how changes in the proteins result in the symptoms of the condition

Question 21

Most diseases (especially non-communicable ones) are the result of the inheritance of gene variants thaf increase the rism if an individual developing a specific disease. What are gene variants?

Answer 21

Different versions of genes, an alternative term for allele

Question 22

In many cases what is it the interaction between that determines the incidence of disease?

Answer 22

The genome and the environment

Question 23

What is the 100 000 genome project?

Answer 23

A UK initiative sequencing the genomes of 100 000 people with cancer and rare genetic diseases. It will provide huge amounts of data showing how particular genotypes combined with environmental conditions raise the risk of or cause specific diseases. The genetic data from patients and their families will be matched with clinical data held on DNA databases. This data will be used by scientisrs to identify mutations associated with different diseases and develop new, targeted therapies

Question 24

What is DNA profiling?

Answer 24

The identification of repeating patterns in the non-coding regions of DNA

Question 25

What is a micro-satellite?

Answer 25

A section of DNA with a 2-6 base sequence repeated 5-100 times

Question 26

What is a mini-satellite?

Answer 26

A section of DNA with a 10-100 base sequence repeated 50 to 100 times

Question 27

Where do the same mini or micro satellites always appear and what is different?

Answer 27

In the same positions on each pair of homogolous chromosomes. However the number of repears of each satellite will vary as different paterns may be inherited from your mother and father

Question 28

Why is the likelihood of two individuals having the same pattern of DNA extremely remote?

Answer 28

There are many different introns and a huge variation in the number of repeats

Question 29

What are restriction endonucleases?

Answer 29

Special enzymes used to chop up strands of DNA at particular points in the intron sequences

Question 30

What are recognition sites?

Answer 30

Specific base sequences where restriction endonucleases cleave the DNA molecules

Question 31

What happens in gel electrophoresis?

Answer 31

The DNA fragments are placed in wells in an agarose gel medium in a buffering solution (to maintain a constant pH) with known DNA fragments to aid identification. The gel contains a fluorescent dye that binds to the DNA fragments in the gel. The dye will fluoresce whe placed under a short-wave ultraviolet (UV) light revealing a band pattern of DNA. This doesn’t bind with the DNA but moves through the gel slightly faster so the cureent can be turned off before the samples run of at the end.

An electric current is passed through the apparatus and the DNA fragments move towards the positive anode because of the negative chatgw or the phosphate groups in the DNA backbone. The fragments move at different rates according to their size and charge. Once the electrophoresis is complete the plate is placed under short wave UV light. The DNA fluoresces and shows up clearly so it can be identified

Question 32

What is southern blotting?

Answer 32

The bame of a process in which DNA fragments are drawn from an electrophoresis gel to a filter leaving the DNA fragments as blots on the filter. The process also denatures the DNA fragments so the strands seperate and the base sequences are exposed

Question 33

What are gene probes?

Answer 33

Short DNA sequences labelled with a fluorescent molecule that are complementary to specific DNA sequences which are being sought

Question 34

What is hybridisation?

Answer 34

The binding of gene probes to the complementary DNA strands

Question 35

How does the process of genetic fingerprinting/ DNA profiling take place?

Answer 35

• Collection: a sample of tissue is taken which contains the DNA to be analysed. This could be blood, cheek cells, semen, hair or any other suitable sample of the individuals cells
• Extraction: the sample is mixed with phenol and chloroform and centrifuged so that the proteins precipitate out. The DNA is then precipitated out of the aqueous water layer using ethanol
• Digestion: restriction enzymes are used to cut the DNA at points close to but not in the minisatellite regions allowing these to keep their original lengths characteristic to the individual being tested
• Separation 1: the digested fragments are then seperated by size by electrophoresis. The gel is then soaked in an alkaline solution to seperate the DNA into single strands. This is denaturation
• Seperation 2 (southern blotting): the DNA is now transferred to a nylon membrane using blotting paper to draw it up out of the gel by capillary action.
• Hybridization: the membrane is immersed in a solution of labelled DNA probed wbicb hybridize with the chosen core sequences. The ultrasound probes are washed off and the membrane is dried
• Develeopment: an X-ray film is placed over the membrane. The film is fogged in he areas where radiation is present, producing an autoradiograph, and the distinctive set of bands known as a DNA fingerprint

Question 36

What are short tandem repeats?

Answer 36

Micro-satellite regions that are now widely used in DNA identification

Question 37

In the microsatellites in the pairs of homogolous chromosomes on a child what does the number of repeats show?

Answer 37

Whether it came from the mother or father