Transcript Section 8 – The human genome project

Unit 1
Cell and Molecular
Biology
Section 8
The human genome project
History



Late 1980’s idea was proposed
Predicted it would take 15 years
Cost about $200 million per year





$1 per base pair
Officially began in 1990
26 June 2000 joint announcement from Blair and
Clinton ‘the draft complete’
Joint publication in Nature and Science 12 Feb 2001
14 Apr 2003 – The finished human genome
Method

Genetic Mapping



Physical Mapping



Identifies relative positions of genes
E.g. Gene 2 lies between genes 1 and 3
Absolute positions of genes on chromosomes
E.g. Gene 2 is 1 million bp from gene 1
DNA sequencing

Actual ATCG combinations
The human genome
The total genetic complement of a cell
Has 3 billion (3 x 109) base pairs
Comprises 23 pairs of chromosomes
How did Biologists go about sequencing
the genome
?
Producing copies by PCR

In order to sequence DNA it is necessary to produce
a huge number of exact copies of the original
stands.

The technique used to do this is known as PCR or
‘Polymerase Chain Reaction’.

Once the copies of DNA have been produced they
can be analysed.

Note – This is the technique used by forensics to
amplify tiny samples of DNA for ‘fingerprinting’
The polymerase Chain
Reaction
DNA duplex (two strands)
5'
3'
3'
5'
denature
5'
3'
primer
primer
3'
5'
5'
3'
Taq
4
synthesis
Taq
3'
5'
etc.
8
16
repeat
cycle
Understanding PCR
1. PCR can amplify any DNA sequence hundreds of millions of
times in just a few hours. It is especially useful because it is
highly specific, easily automated and capable of amplifying
minute amounts of sample
2. The whole process is only possible because of a special heatstable enzyme called Taq polymerase, isolated from
thermophilic bacteria.
3. The enzyme Tac polymerase is able to tolerate temperatures of
95C and has a temperature optimum of 72C.
4. This enzyme can synthesise the complementary strand of a given
DNA strand in a mixture containing the four DNA nucleotide
bases and two short DNA fragments called primers. Each
primer is usually about 20 base pairs (bp) long. The primers are
designed to bind to the DNA at either side of the target
sequence.
Procedure
Step 1 – The DNA is heated to 950 C breaking the
hydrogen bonds and separating the strands.
Step 2 – The strands are cooled to between 55 –
700C and the primers added.
Step 3 – The strands are heated to between 70 – 72
0C so that Taq Polymerase can copy each strand
from the point of the primer.
Summary
PCR requires the following:



Template DNA
Primers – starting points for the construction of new
strands
Taq Polymerase – a polymerase enzyme which
works at high temperatures
Supply of nucleotides
PCR can amplify a single strand of DNA by a factor
of millions
Genome mapping
genetic map - allows relative positions
(a)
II
of heterozygous linked alleles to be located
I
I I I I I I I I
physical map - allows precise location of
(b)
specific DNA sequence to be located.
clone map
(c)
Sequence -Allows sequence of nucleotides to
be determined
AGGTCGCGATGCTA
Genetic linkage mapping
1. Linkage mapping can be used to locate genes on
particular chromosomes and establish the order of these
genes and the approximate distances between them.
2. This idea is based on the fact that the further apart
linked genes are on chromosomes the more likely
crossing over will take place resulting in more
recombinants being formed.
3. The greater the number of recombinants, the further
apart linked genes are on a chromosome. Working out
the number of recombinants relative to the parental
genotypes gives a percentage value which is called the
recombination frequency.
Example:
Linked Gene Pair
Recombination Frequency %
AB
11
AC
7
BC
18
The information from the table can be used to produce a chromosome map.
11 units
B
7 units
A
18 units
C
Genetic Linkage Mapping relies on having
genetic markers that are detectable –
sometimes these are genes that cause
disease, traced in families by pedigree
analysis. The marker alleles must be
heterozygous and be linked on the same
chromosome so that recombination can be
detected.
The overall result of genetic mapping is to
produce a picture of the locations of the marker
loci on the chromosomes – rather like
establishing the order of the cities and large
town between two points on a map.
Physical Mapping
1. Physical mapping is required to add some more of the detail to
what is obtained by genetic mapping
2. As with genetic maps, construction of a physical map requires
markers that can be mapped to a precise location on the DNA
sequence.
3. The distance between markers is usually expressed as a number
of nucleotides in a physical map.
4. A physical map can be made by isolating DNA from a
chromosome then cutting it using restriction enzymes (also
known as restriction endonucleases) to construct a pattern.
5. Different restriction enzymes cut the DNA at
different points as each recognises a particular
short sequence of bases occurring in the DNA.
Where the sequence is recognised, the
enzyme cuts the DNA so that it is cut into
fragments.
6. By using combinations of restriction enzymes
and working out the size of the fragments it is
possible to recognise a pattern. The fragments
can be identified by their size or by using a
specific DNA probe to bind to its
complementary sequence.
Physical restriction mapping example
1.
2.
Genes do not exist as separate entities but
as part of the larger DNA molecule
DNA can be broken up into fragments by
ENDONUCLEASES which cut at specific
base sequences - look up page 316 of your
textbook for more examples
G
G
C
C
CUT FRAGMENT
G
G
C
C
Not 1 - always cuts at the following sequence of eight pairs. This
happens on average every 65536 nucleotide pairs ( 1 in 48 ) and
produces much larger fragments
C C G
C
G C G G
C CC G GC C
G G
G G C G G C G G
C C G
C
C C G

The resulting fragments can be separated using GEL
ELECTROPHORESIS and used to physically map the sections of
DNA - See textbook p317
The following table shows the fragments produced from a 15kbp
fragment
C
14
1
C C G
Bam H1
EcoR1
Pst1
Bam H1
plus
EcoR1
Bam H1
plus
Pst1
EcoR1
plus
Pst1
12
3
8
7
11
3
1
8
6
1
7
5
3
G C

G C G G
G C G G
C C

Bam H1
plus
EcoR1
plus
Pst1
6
5
3
1
ANALYSING THE FRAGMENTS
3
0
9
6
15
12
kbp
A) BAM H1 (14 +1)
14
1
B) EcoR1 (12 +3)
12
3
12
Option 1
3
Option 2
C) BAM H1 plus Eco R1 (11 +3 +1)
3
11
12
1
2
1
Option 1
Option 2
Option 2 for EcoR1 above would give 12 +2 +1 with the double digest which is INCORRECT.
This means that the fragment must have been cut with the following orientation
EcoR1
BAM H1
By repeating this procedure it is possible to build up a RESTRICTION MAP for this
section of DNA - this lets us know the base sequence at each point of cut,
3
0
15
12
9
6
kbp
A) BAM H1 (14 +1)
14
1
B) Pst 1 (8 +7)
8
7
Option 1
8
7
Option 2
C) BAM H1 plus Pst1 (8 +6 +1)
8
6
1
7
7
1
Option 1
Option 2
Option 2 for EcoR1 above would give 7 +7 +1 with the double digest which is INCORRECT.
This means that the fragment must have been cut with the following orientation
EcoR1
Pst 1
BAM H1
Checking the results of the triple digest EcoR1 plus Pst1 plus Bam H1 show that are
map is correct.
•Since each cut with a known enzyme is a specific base sequence comparing restriction
maps allows biologists to look for the numbers and locations of these base sequences.
The theory is that the greater the number of sequences and the closer their location on
the DNA the more closely related the individuals
In the following example three endonucleases have been used (1-3) and have cut the strands
at the points shown. The reults indicate that individuals A and B are more closely related
that individuals A and C or B and C
A
1
2
3
B
1
2
3
C
1
3
2
Physical Mapping relies on the availability of many copies of the DNA fragment.
This is only possible because of the technique known as POLYMERASE CHAIN
REACTION or PCR which allows many copies of the section of DNA to be produced
DNA sequencing
1. The final stage of the genome project is to determine and assemble
the actual DNA sequence itself.
2. There are several critical requirements for this part:a. Single stranded DNA fragments must be generated as the
templates;
b. sequencing technology must be accurate and fast;
c. computer hardware and software must be available to analyse
the sequence data.
3. The technique used for sequencing is called dideoxy chaintermination method.
4. This method relies on making a copy of the DNA template to be
sequenced using:a. a DNA polymerase;
b. a primer;
c. the four dNTPs (deoxyribonucleoside triphosphates dATP,
dCTP, dTTP and dGTP) to extend the chain;
d. a labelled dNTP; nowadays using a fluorescent dye rather
than a radioactive element as used in the past.
1. In the correct conditions the polymerase can make a
copy of the DNA by a process that is essentially the
same as that used in DNA replication.
2. The chain termination part is what makes the key
difference from normal DNA replication. This involves
setting up four separate reactions, each including one of
the four dideoxy NTPs (ddATP, ddGTP, ddCTP and
ddTTP).
3. These modified nucleotides cannot form the next
phosphodiester bond in the growing chain – hence when
a ddNTP is incorporated into the copy, it terminates the
process.
4. The large number of fragments that are produced in the
four reactions produce a set of sequences that differ in
length by one base, and end with a particular ddNTP.
deoxyribonucleoside
triphosphate
Allows strand extension at
3’ end
dideoxyribonucleoside
triphosphate
Prevents strand extension at 3’
end
Normal
deoxyribonucleoside
triphosphate precursors
(dATP, dCTP,dGTP, and
dTTP)
OLIGONUCLEOTIDE primer
for DNA polymerase
Small amount of one
dideoxyribonucleoside
A
Rare incorporation of
dideoxyribonucleoside by DNA
polymerase blocks further growth
of the DNA molecule
5’
3’
GCTACCTGCATGGA
CGATGGACGTACCTCTGAAGCG
5’
single-stranded DNA molecule
to be sequenced
The fragments can be separated using gel electrophoresis -to see animated
version of this go ‘DNA sequencing by enzyme methods’ click here
Separating DNA fragments:
DNA fragments can be separated by gel
electrophoresis
Largest fragments
Smallest fragments
+
gel with DNA
fragments
DNA moves to
the positive terminal
due to it’s overall
negative charge
DNA
sequencing:
separating
the DNA
fragments
The different fluorescent
labels in the copied DNA
strand are detected as
they come off the bottom
of the gel.
Automated DNA sequencing
This gives a direct
readout of the
sequence and the
process can be
automated so that
it is much faster
than by
conventional
sequencing.
A C T G A C
(d)
gel
(c)
(a)
(b)
To see an animated version of this process go
to the ‘cycle sequencing’ section on the
following website of the Biology Animation
Library

http://www.dnalc.org/resources/BiologyAnima
tionLibrary.htm
Activity

Look at the arrangements document to clarify what
information is required.

Read DART pg 73 – 81.

Read the Monograph pg 67 – 79

Scholar – 8

Internet research