Topic 7 - Modern Genetics: Using gene sequencing

Question 1

number of genes in a human genome

Answer 1

20000

Question 2

number of base pairs in human genome

Answer 2

3100000000

Question 3

What is a genome?

Answer 3

all the genes in a cell or organism

Question 4

bases only account for ____ % of the genome?

Answer 4

1.5%

Question 5

if bases only account for 1.5 % of the genome, then what makes up the other 89.5% ?

Answer 5

non-coding DNA

Question 6

what does non-coding DNA not do?
and what does it do?

Answer 6

• doesn’t code for amino acids
• makes DNA work properly
• can be used by scientists

Question 7

what does Vitro mean?

Answer 7

outside of a living organism
- fertilisation in culture dish, outide of female body

Question 8

What does Vivo mean?

Answer 8

inside of a living organism

Question 9

example of In Vitro

Answer 9

IVF
In Vitro Fertilisation
- fertilisation in culture dish, outide of female body

Question 10

what does IVF stand for?

Answer 10

In Vitro Fertilisation

Question 11

what does PCR stand for

Answer 11

Polymerase Chain Reaction

Question 12

Whatis PCR

Answer 12

its in in vitro method used to amplify (make more of) DNA fragments

Question 13

PCR

what does DNA polymerase do?

Answer 13

• assembles the DNA nucleotides to form newly synthesised DNA

Question 14

What does the PCR method involve? and what does this try and replicate?

Answer 14

cyclical cooling and heating to a series of temps
- tries to mimic the events of DNA replication in Interphase cell cycle

Question 15

what are the 3 key temperatures for PCR testing?

Answer 15

• 72°C
• 95°C
• 50°C -65°C

Question 16

what is thermophilus aquaticus?
(it has TAQ Polymerase)

Answer 16

its a type of bacteria that has TAQ polymerase, an enzyme that’s stable at high temperatures such as 95°C
- its magnesium dependant, which is why it needs magnesium ions

Question 17

how many pcr stages are there?

Answer 17

6

Question 18

PCR stages:

Stage1

Answer 18

• reaction mixture set up, containing DNA sample, mg ions, primers, free nucleotides, and DNA polymerase

Question 19

PCR stages:

Stage 2

Answer 19

• mixture heated to 95°C
• • this breaks hydrogen bonds btwn the 2 \dna strands, resulting in single strands

Question 20

why can we heat the mixture to 95°C without denaturing anything?
dependant on anything, how do we compromise

Answer 20

• TAQ polymerase is an enzynme stable at high temperatures and so doesnt denature at 95°C.
• but its mg dependant wich is why we use mg ions

Question 21

PCR stages:

Stage 3
annealing (to join) - refers to the pairing of complementary DNA (or RNA) sequences by hydrogen bonding

Answer 21

• mixtue is cooled to btwn 50°C and 65°C
• at this temp, the primers will anneal to their complementary base sequence at 3’ end of each single strand of DNA

Question 22

PCR stages

what are primers?

Answer 22

short pieces of single-stranded DNA that are complementary to the target sequence

Question 23

PCR stages:

Stage 4

Answer 23

• TAQ polymerase binds to the small section of DNA and the temp is increased to 72°C (optimum for this enzyme)

Question 24

PCR stages:

Stage 5

Answer 24

• The enxyme moves along in the 5’ to 3’ direction, catalysing condensation reactions to join free DNA nucleotides by phosphodiester bonds.

Question 25

PCR stages:

Stage 6

Answer 25

• at the end of the first cycle, it will result ion 2 identical copies of the DNA sample
• as PCR is a cyclical reaction, it can be repeated - eventually creating a billion copies of the DNA sample

Question 26

what is DNA sequencing?
who and when

Answer 26

• processs of working out the order of bases in a strand of DNA
• Fredrick Sanger 1980

Question 27

DNA sequencing

terminator bases?
is what?
how many per base?

Answer 27

• a special nucleotide
• like a stop codon but only for 1 nucleotide unlike a triplet
• one for each of the 4 bases in DNA

Question 28

DNA sequencing

what do terminator bases lack?
and what does this mean?
and what does that then do/terminate?

Answer 28

• an -OH grp on C3of deoxyribose, therefore it cannot bond to the next phosphate grp on the following nucleotide, as no phosphodiester bond can form
• therefore it terminates strand extension during PCR

Question 29

DNA profiling/fingerprinting produces what?

Answer 29

• produces a specific pattern of DNA bands from an individual’s genome

Question 30

DNA profiling/fingerprinting

invented when?
- used for what?

Answer 30

1984
- check relations (paternity tests), for crimes

Question 31

DNA profiling/fingerprinting

do they use exons (coding) or introns (non-coding) regions

Answer 31

• use introns as extrons already code of sum

Question 32

DNA profiling/fingerprinting

what are satellites?

Answer 32

• short sections of repeated code in introns

Question 33

DNA profiling/fingerprinting

2 types of satellites:

Answer 33

• mini-satellites
• micro-satellites

Question 34

DNA profiling/fingerprinting

mini-satellites?

Answer 34

• 10-100 base sequences will be repeated 50 to 100 times

number of times these repeats happen are hugely varieable btwn individuals, so this is what we want to isolate and analyse

Question 35

DNA profiling/fingerprinting

micro-satellites?

Answer 35

• 2-6 base sequences will be repeated 5-100 times

number of times these repeats happen are hugely varieable btwn individuals, so this is what we want to isolate and analyse

Question 36

VNTRs

Answer 36

Variable Number Tandem Repeat

Question 37

DNA profiling/fingerprinting
Stages:

Answer 37

• isolate these sections of DNA using restriction endonucleases (enxymes)
• amplify using PCR
• organise into size
• • then use gel electrophoresis

Question 38

restriction endonucleases
(enxyme)
does what?

Answer 38

• type of enzyme that cuts up DNA at specific sequence of bases called recognition sites.

recognition sites: sequence of nucleotides

Question 39

What are recognition sites?

Answer 39

recognition sites: sequence of nucleotides

Question 40

how does alkali break down DNA

Answer 40

• denatures DNA, breaks down hydrogen bonds -> ends up with seperate strands

Question 41

Southern blotting with DNA probles