Download Sequencing a genome

Title: Studying whole genomes
Homework: learning package 14 for Thursday
23 May 2017
Aims from specification
(a) outline the steps involved in sequencing the
genome of an organism;
(b) outline how gene sequencing allows for
genome-wide comparisons between individuals
and between species (HSW7b);
(c) define the term recombinant DNA;
Genomes
• 1950’s
– Learnt that DNA is the genetic material
• Gene technology
– Use of DNA to produce something that we want
• Developing rapidly
• Becoming more and more able to alter genes within
organisms
• Point to think about
– Just because we can do something does that mean that we
should do it?
Manipulating DNA
• Advances in DNA technology
– DNA profiling (genetic fingerprinting)
– Genomic sequencing
– Comparative genome mapping
– Genetic engineering
– Gene therapy
Genome
• All the genes possessed by an individual
organism, or a population of organisms.
• The whole sequence of bases in all of the DNA
in an organism.
Human Genome Project
• 1988
– International project started to discover the sequence of
bases in each of the 23 different types of chromosomes
found in human cells
• 2000
– A working draft sequence was produced
Facts about human genome
• 99.9% of the base sequence in our DNA seems to be
identical in all humans
• Variation is caused by the variable 0.1%
– This 0.1% is very variable
– Variations can be used for DNA profiling
• 2% of human genome codes for the manufacture of
proteins
– Giving around 20 000 genes in the human genome (even mice
have more!!)
• The rest of the “junk” genome, may be involved in gene
expression
Chain Termination Method of DNA Sequencing
Sequencing a genome
• The chain termination method is used to
determine the order of bases in a section of DNA
• Make multiple labelled copies of each small
length of DNA
– Lengths of DNA mixed with
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DNA Polymerase
Primer
“normal” DNA nucleotides
“labelled” DNA nucleotides
– dideoxy nucleotides
– Four colours of dye used for bases A, T, G and C
– If incorporated in nucleotide chain – chain stops growing
Sequencing a genome
• Result
– Many different chains of different lengths
– Each length ends with a labelled nucleotide
• Mixture of lengths of DNA separated using
electrophoresis
– The shorter the length of DNA the faster it travels
• Computer records the colours as they pass the end of
the tube, if there are enough fragments then every
base in the complete chain will be represented.
• Computer works out the sequence of the length of
DNA
Sequencing a genome
• Process is largely automated
– Put your DNA sample into a sequencing machine
– Get a print out from the bottom
• Preparation of DNA and analysis is still time
consuming
Sequencing a genome
• Chain-Termination Method can only be used for DNA
fragments up to 750bp long
• Therefore, the entire genome is broken up and sequenced
in sections
• Sequencing is carried out on overlapping regions
• Stages
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Genome mapping
Mechanically break into smaller sections
Carry out sequencing on overlapping sections
Analyse and put back together to form the complete code
Whole genome sequencing using BACs
1. Mapping the genome
• Genomes are mapped to identify which chromosome or
section of chromosome it came from
• Uses previous information to help find the location –
microsatellites are of use here.
• Microsatellites are short sections of DNA (3-4bps) that
repeat over thousands of locations on the genome
• Samples of genome are mechanically broken into smaller
sections ~100,000bps
• This is sometimes referred to as the “shotgun” approach.
2. BACs
• Sections of DNA placed into bacterial artificial
chromosomes (BACs) and transferred to E.
Coli cells.
• Cells grow in culture, many copies (clones) are
produced
• These cells are referred to as clone libraries
3. Sequencing BACs
• Cells containing specific BACs are taken and cultured. DNA
is extracted using restriction enzymes to cut it up into
smaller fragments
• Different restriction enzymes are used to give different
fragment types (will “cut” DNA at different places)
• Fragments separated using gel electrophoresis
• Fragment sequencing using an automated process
• Computer programmes compare overlapping regions from
the cuts made by different restriction enzymes in order to
reassemble the whole BAC segment sequence.
Exam questions
1. To sequence a small DNA fragment, a singlestranded DNA template and DNA polymerase are
needed.
A. Name the other three reactants needed for a
sequencing reaction (3)
B. Describe and explain the process of sequencing a
small DNA fragment (6)
2. The genomes of over 200 different species have
been sequenced. Describe how a genome can be
sequenced using BACs (8)
Answers 1A
1. DNA primer
2. Free nucleotides
3. Fluorescently-labelled modified nucleotides
Answers 1B
1.
2.
3.
4.
5.
6.
The reaction mixture is added to four tubes, with a different modified
nucleotide in each tube
The tubes undergo PCR to produce lots of strands of DNA of different
lengths
Each strand of DNA is a different length because each one terminates at
a different point depending on where the modified nucleotide was
added
The DNA fragment in each tube are separated by electrophoresis and
visualised under UV light
The smallest nucleotide is at the bottom of the gel and each band after
this represents one more base added
So the bands can read from the bottom of the gel to the top, forming the
base sequence of the DNA fragment
Answers 2
1.
2.
3.
4.
5.
6.
7.
8.
The genome is cut up into smaller fragments using restriction enzymes
The individual fragments are inserted into bacterial artificial
chromosomes/BACs, which are inserted into bacteria
Each BAC contains a different DNA fragment, so each bacterium contains
a BAC with a different DNA fragment.
The bacteria divide, creating colonies of cloned cells that contain their
specific DNA fragment
Together the different colonies make a complete genomic DNA library
DNA is extracted from each colony and cut up using restriction enzymes,
producing overlapping pieces of DNA
Each piece of DNA is sequence, using the chain termination method, and
the pieces are put back in order to give the full sequence from the BAC
Finally the DNA fragment from each different BAC is put back in order
using computers, to compete the entire genome
Answers 1B
1. DNA is mixed with free nucleotides, primers and DNA
polymerase
2. The mixture is heated to 95oC to break the hydrogen bonds
3. The mixture is then cooled to between 50-65oC to allow the
primers to bind/anneal to the DNA
4. The primers bind/anneal to the DNA because they have a
sequence that is complementary to the sequence at the
start of the DNA fragment
5. The mixture is then heated to 72oC and DNA polymerase
lines up free nucleotides along each template strand,
producing new strands of DNA
6. The cycle would be repeated over and over to produce lots
of copies