DNA sequencing Flashcards
1
Q
DNA Sequencing
A
- Cloning technology leads to DNA sequencing
- Sequencing is the process of determining the exact nucleotide sequence of a DNA molecule
- Most DNA sequencing is based on the sanger chain termination method
2
Q
who developed Sanger sequencing
A
- Developed by fred sanger and colleagues in 1977
3
Q
for a fragment to be sequenced it must first be..
A
replicated
4
Q
what gives a pure sample of fragment of interest
A
cloned in a vector or amplified by PCR
5
Q
sanger sequencing
A
- DNA synthesis complementary to the strand of interest
- Random incorporation of nucleotides which terminate DNA chain
- These ddNTPs lack an OH group for the next nucleotide to attach
- DNA synthesis with all the possible positions of ddNTPs until the entire sequence is known
- Strands of many different lengths are created
6
Q
what do the fluorescent tags allow
A
whole reaction (all ddNTPs) to be done in one tube - The different length fragments are separated on polyacrylamide gel with 1bp resolution
7
Q
what happens to the different length fragment
A
separated on polyacrylamide gel with 1bp resolution
8
Q
autoradiography
A
- Sequence of new strands is read and sequence of template is deduced
- Because so many copies are made and the ddNTPs are incorporated randomly, you eventually send up a mixture of DNA molecules that stop at every single letter
9
Q
for the chain termination method you need:
A
- single- stranded DNA template
- a DNA primer
- DNA polymerase
- Normal deoxynucleotide triphosphates (dNTPs)
- Modified di-deoxynucleotide triphosphates (ddNTPs) which terminate DNA strand elongation
10
Q
forensic applications of DNA sequencing
A
- Mainly applied to the analysis of mitochondrial DNA (mtDNA)
- Mitochondria contain a small circular genome, 37 genes
- Non-coding control region exhibits a lot of variation between individuals therefore ideal for forensics
11
Q
mtDNA
A
- Hypervariable regions HVI and HVII contain nucleotide sequence variation
- Maternally inherited
- Traditionally used for degraded samples due to number of copies per cell
12
Q
what is the human genome project
A
- ‘ an international research effort to sequence and map all of the genes- together known as the genome- of members of our species, homosapiens’
13
Q
human genome project
A
- Used sanger sequencing to sequence the entire genome
- Started in 1990-2003
- Culmination of 50 years research in molecular biology
- Discovered that the genome has around 20,500 genes and mapped genes
14
Q
Next generation sequencing
A
- Massively parallel approach to sequencing taking over from sanger sequencing
- Cheaper, quicker, smaller sample needed and ultimately more accurate
- Chemical reaction and signal detection combined
15
Q
NGS is a broad term for several different approaches to sequencing:
A
- Illumine sequencing
- Ion torrent
- AB SOLiD
- DNA immobilised on chip/bead and repeatedly interrogated
