DNA sequencing

Question 1

what does cloning technology lead to?

Answer 1

DNA sequencing

Question 2

what is sequencing

Answer 2

process of determining the exact nucleotide sequence of a DNA molecule

Question 3

what is most DNA sequencing based on

Answer 3

Sanger chain termination method

Question 4

who was the Sanger sequencing developed by and when?

Answer 4

Fred Sanger 1977

Question 5

what must happen to a fragment before it is sequenced?

Answer 5

replicated

Question 6

how is a pure sample of fragment obtained?

Answer 6

cloned in a vector or amplified by PCR

Question 7

Sanger sequencing

Answer 7

DNA synthesis complementary to the strand of interest

Random incorporation of nucleotides which terminate DNA chain

These ddNTPs lack OH group for the next nucleotide to attach

DNA synthesised with all the possible positions of ddNTPs until the entire sequence is known

Strands of many different lengths are created

Question 8

what are fluorescent tags for?

Answer 8

allow whole reaction to be done in one tube

Question 9

how are the different length fragments separated?

Answer 9

on polyacrylamide gel with 1bp resolution

Question 10

autoradiograph

Answer 10

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 with a mixture of DNA molecules that stop at every single letter.

Question 11

what 5 things are needed for the chain termination method

Answer 11

Single stranded DNA template

a DNA primer

a DNA polymerase

normal deoxynucleotide triphosphates (dNTPs)

modified di-deoxynucleotide triphosphates (ddNTPs) which terminate DNA strand elongation

Question 12

forensic applications of DNA sequencing

Answer 12

mainly applied to 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

Question 13

what is mtDNA

Answer 13

hypervariable regions HVI & HVII contain nucleotide sequence variation

maternally inherited

traditionally used for degraded samples due to number of copies per cell

Question 14

what is the human genome project

Answer 14

“an international research effort to sequence and map all of the genes – together known as the genome – of members of our species, Homo sapiens”

Used Sanger sequencing to sequence the entire human genome

Question 15

when did the human genome project start and finish

Answer 15

Started in 1990, completed in April 2003

Question 16

how many years of molecular biology research were used in the human genome project

Answer 16

50

Question 17

how many genes were discovered to be in the genome?

Answer 17

20,500

Question 18

what is next generation sequencing

Answer 18

massively parallel approach to sequencing

chemical reaction and signal detection combined

hundreds of thousands of DNA fragments simultaneously sequenced

Question 19

advantages if next generation sequencing

Answer 19

cheaper
quicker
smaller sample needed
more accurate

Question 20

next generation sequencing is a broad term for what 5 different approaches

Answer 20

Illumine sequencing

Roche 454 sequencing

Ion torrent

AB SOLiD

DNA immobilised on chip/bead and repeatedly interrogated

Question 21

forensic applications of NGS

Answer 21

STRs – can examine both allele size and sequence, useful for mixed DNA samples

Can carry out various tests simultaneously on a sample: STRs, mtDNA, SNPs

One machine, one software

Large amount of data. Bioinformatics (multidisciplinary field)

Pharmacogenomics – genetic differences in metabolic pathways

Molecular autopsy (post-mortem genetic testing)