Topic 7 - Modern Genetics: Using gene sequencing Flashcards
number of genes in a human genome
20000
number of base pairs in human genome
3100000000
What is a genome?
all the genes in a cell or organism
bases only account for ____ % of the genome?
1.5%
if bases only account for 1.5 % of the genome, then what makes up the other 89.5% ?
non-coding DNA
what does non-coding DNA not do?
and what does it do?
- doesn’t code for amino acids
- makes DNA work properly
- can be used by scientists
what does Vitro mean?
outside of a living organism
- fertilisation in culture dish, outide of female body
What does Vivo mean?
inside of a living organism
example of In Vitro
IVF
In Vitro Fertilisation
- fertilisation in culture dish, outide of female body
what does IVF stand for?
In Vitro Fertilisation
what does PCR stand for
Polymerase Chain Reaction
Whatis PCR
its in in vitro method used to amplify (make more of) DNA fragments
PCR
what does DNA polymerase do?
- assembles the DNA nucleotides to form newly synthesised DNA
What does the PCR method involve? and what does this try and replicate?
cyclical cooling and heating to a series of temps
- tries to mimic the events of DNA replication in Interphase cell cycle
what are the 3 key temperatures for PCR testing?
- 72°C
- 95°C
- 50°C -65°C
what is thermophilus aquaticus?
(it has TAQ Polymerase)
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
how many pcr stages are there?
6
PCR stages:
Stage1
- reaction mixture set up, containing DNA sample, mg ions, primers, free nucleotides, and DNA polymerase
PCR stages:
Stage 2
- mixture heated to 95°C
- this breaks hydrogen bonds btwn the 2 \dna strands, resulting in single strands
why can we heat the mixture to 95°C without denaturing anything?
dependant on anything, how do we compromise
- 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
PCR stages:
Stage 3
annealing (to join) - refers to the pairing of complementary DNA (or RNA) sequences by hydrogen bonding
- 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
PCR stages
what are primers?
short pieces of single-stranded DNA that are complementary to the target sequence
PCR stages:
Stage 4
- TAQ polymerase binds to the small section of DNA and the temp is increased to 72°C (optimum for this enzyme)
PCR stages:
Stage 5
- The enxyme moves along in the 5’ to 3’ direction, catalysing condensation reactions to join free DNA nucleotides by phosphodiester bonds.
PCR stages:
Stage 6
- 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
what is DNA sequencing?
who and when
- processs of working out the order of bases in a strand of DNA
- Fredrick Sanger 1980
DNA sequencing
terminator bases?
is what?
how many per base?
- a special nucleotide
- like a stop codon but only for 1 nucleotide unlike a triplet
- one for each of the 4 bases in DNA
DNA sequencing
what do terminator bases lack?
and what does this mean?
and what does that then do/terminate?
- 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
DNA profiling/fingerprinting produces what?
- produces a specific pattern of DNA bands from an individual’s genome
DNA profiling/fingerprinting
invented when?
- used for what?
1984
- check relations (paternity tests), for crimes
DNA profiling/fingerprinting
do they use exons (coding) or introns (non-coding) regions
- use introns as extrons already code of sum
DNA profiling/fingerprinting
what are satellites?
- short sections of repeated code in introns
DNA profiling/fingerprinting
2 types of satellites:
- mini-satellites
- micro-satellites
DNA profiling/fingerprinting
mini-satellites?
- 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
DNA profiling/fingerprinting
micro-satellites?
- 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
VNTRs
Variable Number Tandem Repeat
DNA profiling/fingerprinting
Stages:
- isolate these sections of DNA using restriction endonucleases (enxymes)
- amplify using PCR
- organise into size
- then use gel electrophoresis
restriction endonucleases
(enxyme)
does what?
- type of enzyme that cuts up DNA at specific sequence of bases called recognition sites.
recognition sites: sequence of nucleotides
What are recognition sites?
recognition sites: sequence of nucleotides
how does alkali break down DNA
- denatures DNA, breaks down hydrogen bonds -> ends up with seperate strands
Southern blotting with DNA probles
