Topic 7.1- Gene sequencing

Question 1

Define genome

Answer 1

the complete set of genetic information contained in the cells of an organism

Question 2

What is DNA/gene sequencing?

Answer 2

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

Question 3

How can we amplify DNA sequences in order to sequence them?

Answer 3

PCR (polymerase chain reaction)

Question 4

Describe the reaction mixture in the first stage of PCR

Answer 4

• DNA fragments to be amplified
• complimentary primers
• free nucleotides
• Taq DNA polymerase

Question 5

Summarise the process of amplifying DNA fragments using PCR

Answer 5

1. Heated to 90-95 degrees for 30s to denature and break apart H bonds in the DNA strands
2. Cooled to 50-55 degrees for 20s to allow primers to bind/anneal
3. Heated again to 72 degrees for at least 1 minute to activate DNA polymerase and allow free nucleotides to bind
4. New DNA acts as a template for next cycle

Question 6

How can DNA sequencing be used in medicine?

Answer 6

• to screen for heritable conditions
• when the base sequence of a particular gene or allele is known, a person’s DNA can be tested to see if that gene or allele is present

Question 7

Describe the process of DNA sequencing

Answer 7

1. DNA strands chopped into smaller pieces
2. double strands separated into single strands
3. PCR used to replicate the DNA fragments and produce large quantities for analysis
4. labelled terminator bases added to the single strands of DNA
5. coloured tags on the terminator bases enable the sequence of bases to be read rapidly by an automated system

Question 8

Define terminator bases

Answer 8

modified versions of the 4 nucleotide bases that halt the production of a DNA molecule as soon as they are incorporated so no new bases can be added

Question 9

What is massively parallel sequencing?

Answer 9

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

Question 10

What are gene variants?

Answer 10

different versions of a gene, alternative term for an allele

Question 11

Define DNA profiling

Answer 11

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

Question 12

What are micro-satellites?

Answer 12

a section of DNA with 2-6 base sequences repeated between 5 and 100 times

Question 13

What are mini-satellites?

Answer 13

a section of DNA with 10-100 base sequences repeated 50 to several hundred times

Question 14

Define the purpose of restriction endonucleases

Answer 14

special enzymes used to chop up DNA strands at different points in the intron sequences

Question 15

What are recognition sites?

Answer 15

specific base sequences where restriction endonucleases chop the DNA molecule

Question 16

Define Southern blotting

Answer 16

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 separate and the base sequences exposed

Question 17

Describe the process of gel electrophoresis

Answer 17

1. DNA fragments placed in wells in an agarose gel medium in a buffering solution with known DNA fragments, to aid identification
2. gel contains a dye which binds the fragments to the gel and also becomes fluorescent when placed under UV, revealing a band pattern of DNA
3. visible dye added that doesn’t bind to the DNA
4. electric current applied and fragments move towards the anode
5. DNA fragments move at different rates according to their size and charge
6. once completed, placed under UV light so the DNA fluoresces

Question 18

Why is gel electrophoresis carried out in a buffering solution?

Answer 18

to maintain a constant pH

Question 19

What is the purpose of adding a non-binding visible dye to electrophoresis?

Answer 19

the dye moves through the gel slightly faster than the DNA fragments so that the current can be turned off before all the samples run off the end

Question 20

What are gene probes?

Answer 20

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

Question 21

What is hybridisation?

Answer 21

the binding of gene probes to the complementary DNA strands

Question 22

What are short tandem repeats?

Answer 22

micro-satellite regions widely used in DNA identification

Question 23

Define polymerase chain reaction (PCR)

Answer 23

the reaction used to amplify a sample of DNA, to make more copies of it very rapidly

Question 24

Define introns

Answer 24

large, non-coding regions of DNA that are removed before mRNA is translated into proteins

Question 25

Define exons

Answer 25

the coding regions of DNA (the genes)

Question 26

How is DNA sequencing used to determine the protein produced from a particular gene?

Answer 26

• universal code means we recognise start and stop codons in a gene
• analysing the base pairs enables us to work out which amino acids will be joined together

Question 27

Give 3 ways in which DNA sequencing is moving scientific knowledge and understanding forward

Answer 27

• disease management: understanding genetic basis of non-communicable diseases, sequencing the genomes of pathogens to identify them and identify the most effective antibiotic
• DNA barcoding: for identifying species and working out evolutionary relationships
• analysing gene expression: knowing the DNA sequence of genes and the proteins they produce