Cloning into Plasmid

wirelessthrillΒιοτεχνολογία

11 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

240 εμφανίσεις


2.7.
3

1

State two examples of the current uses of genetic engineering in agriculture
and/or pharmacy.




Improved crops and animal breeds by increased disease
resistance, heavy metal tolerance and better yield can be
mentioned. Bacteria can be made to m
anufacture useful
compounds for various purposes, for example insulin.

It is an attractive teaching point to emphasise the concentrated
time scale in the development of genetic manipulation
techniques.

1953 Watson and Crick DNA model

1970 first restrictio
n enzymes

1973 plasmid splicing

1977 first engineered bacteria

1985 genetic fingerprinting

1989 cystic fibrosis gene cloned and sequenced

1994 genetically manipulated organisms for food



Two products of recombinent DNA technology include


Insulin (pharma
ceutical)

Genetically modified foods (agriculture)



2.7.
4

3

Explain one potential harmful result of genetic engineering.




Some controversial gene transfers are regarded as harmful, by
some people (eg. bovine somatotropin). A possible problem
exists wit
h the release of genetically engineered organisms in the
environment. They could spread and compete with the naturally
occurring varieties. Some of the engineered genes could also
cross species barriers, and effect the food chain.



2.7.
5

1

State that PCR

(polymerase chain reaction) copies and amplifies minute
quantities of nucleic acid.

PCR is a method which copies tiny amounts of DNA and amplifies it so there is a large amount which
can then be analysed.



2.7.
6

1

State that gel electrophoresis involves

the separation of fragmented pieces of
DNA according to their charge and size.

Restriction endonucleases are used to chop up
DNA into fragments. It will chop different
peoples DNA in different places as every ones
DNA is different, resulting in differe
nt sized
fragments. In gel electrophoresis shown to the
left (with the gel in blue) a drop of these DNA
fragments is placed on the gel and a current
passed through. The fragments are sorted
according to



Size


big molecules travel through
gel slower as
there is more
resistance



Charge


positively charged
particles migrate to the cathode and
negatively charged to the anode.

2.7.
7

1

State that gel electrophoresis of DNA is used in DNA profiling.

2.7.8

2

Describe two applications of DNA profiling.




Appl
ications could include some criminal investigation such as
murder or rape, or paternity suits. The identification of people
that died a long time ago, or information about them is possible
(eg., the dead Tsars of Russia and some Egyptian mummies).

Draw att
ention to the problems of contamination of samples.

DNA in humans is similar, but peoples DNA differs due to mutations resulting in different
sequences and therefore DNA is cut at different places in different people, making it unique.
DNA profiling uses
gel electrophoresis to separate fragments (choppped by restriction
endonucleases) into charge and size. Common uses of DNA profiling are the identification of
parents, and identifying criminals. A small amount of DNA is needed as the pllymerars chain
rea
ction can increase the amount of DNA making enough for gel
-
electrophoresis.


Paternity


Inheritance of bands

Each child receives one band from their mother, and one from their father. The first
step in interpreting this autorad is to identify which bands
were contributed by the
mother. Then the remaining bands can be compared to the alleged father to determine
if he is included or excluded.




Results from a single locus probe DNA fingerprint analysis for a man and
woman and their four children are shown in the DNA profile. Which child
is least likely to be the biological offspring of this couple?



All of the children have 1 ba
nd from the mother and 1 band from the
father, with the exception of child 2. Child 2 appears to share two bands
with the mother and none from the "father". Obviously, the mother could
not have contributed both bands to her child.

There are several possi
ble explanations for this banding pattern:

The band contributed by the biological father just
happened to match one of the mother's bands.

Child 2 is actually the "mother's" sister.

An error was made in loading the DNA samples into the
lanes and the mot
her's DNA was accidently loaded into
lane 2.


DNA profile analysis


rape investigation


DNA evidence is most powerful when used to demonstrate that an individual is not
the source of biological evidence in a criminal investigation. In the case of a rape
, if
the male fraction of the DNA evidence contains alleles that are not shared with the
suspect, then the suspect could not have deposited that sperm. The suspect would be
excluded as a possible source of the DNA found during the sex assault exam.


Noti
ce that in this set of evidence, the victim's DNA profile
serves as an internal control. The victim's known blood
sample (lane 1) should, and does, match the female fraction of
the DNA from the sex assault exam (lane 4).

In contrast, the defendant's known

blood sample (in lane 2)
does not match the male fraction of the evidentiary DNA (in
lane 5). All it takes to exclude a defendant is a single band in
the evidentiary lane that is not found in the defendant's lane.
In this case, the defendant does not have

the top band in lane
5, thus he could not have been the source of the DNA in lane
5.











2.7.
9

2

Outline the process of gene therapy using a named example.




This involves replacement of defective genes. White blood cells
or bone marrow cells are

removed and, by means of a vector, the
normal gene is introduced and inserted into the chromosome.
The cells are replaced in the patient so that the normal gene can
be expressed. Examples are the use in cystic fibrosis and SCID (a
condition of immune defi
ciency, where the replaced gene allows
for the production of the enzyme ADA
-

adenosine deaminase).
A cure for thalassaemia is also possible.

Gene therapy


Genetic engineering can be used to correct some genetic disorders in individuals. This
correction
enables the recessive faulty gene to be masked by a dominant normal gene so the
individual’s normal gene is expressed Genes are controlled by alleles on the DNA.


Gene therapy is best suited to the treatment of single enzyme deficience disease where
introd
ucing the normal allele will cause the normal allele to be expressed, and therfore the
enzyme made.


EG


disease caused by lack of enzyme ADA Adenosine deaminase.


To treat the disease


a)

the cloned gene is inserted into a retrovirus by using recombinent D
NA technology.
The retrovirus inserts a copy of its nucleic acid into the chromosomal DNA of the
host cell

b)

The retrovirus infects the cellsfrom the bone marrow which have been removed from
the patient and cultured

c)

The viral DNA with the normal allele is i
nserted into the chromosome.

d)

The cell replicates by mitosis producing identical daughter cells throughout life. This
type of gene therapy is called somatic gene therapy as it only changes the normal
body cells, not the sex cells so the disorder could sti
ll be passed on to offspring.





S
S
C


Topic 3
-

Genetics


Introduction:

Teachers may wish to introduce the topic by referring to Brno
(Czech Republic) and the historical

origins of the science of
Genetics in the work of Mendel (3.3.5
-

3.3.6). Another way is to
start with examples of modern genetics in human health, plant
breeding and animal husbandry. The topic allows for the teaching
of the principles of genetics includ
ing the relationship between
genes and alleles and chromosome movement. The classical
techniques of solving genetics problems can be covered in 3.4.10
and 3.4.11. Note, however, that students are not expected to be
familiar with dihybrid crosses in this pa
rt of the programme.

A.S.

Obj

3.1

Chromosomes, genes and alleles

(1h)

3.1.1

1

State that an eukaryote chromosome is made of DNA and protein.




The names of the proteins ar
e not required, nor is the
structural relationship between the DNA and the
proteins.


The eukaryote chromosome is made up of deoxyribonucleic acid (DNA) wound around beads of
proteins.

3.1.2

1

State that chromosomes can be stained to show banding.




Th
e staining technique is not required.


Staining techniques can produce band patterns on the chromosomes, which along with the size of the
chromosomes can help in recognising chromosomes and pairing homologous (identical but one from
mother and one from fa
ther) together. There are 22 identical pairs and 2 sex chromosomes in humans,
making a total of 46 chromosomes. Below is the chromosomes before they are arranged.






The homologous pairs are lined up from biggest to smallest with sexcells at the end. This arrangement
is called a in a karyotype. Karyotyping can be done by using enlarged photocopies of chromosomes.


3.1.3

1

Stat
e that the chromosome structure and banding can be used to arrange the
chromosomes in their pairs.




Karyotyping can be done by using enlarged
photocopies of chromosomes.

3.1.4

2

Describe one application of karyotyping (cross reference 3.3.4).


The fol
lowing
karyotype

is taken from a normal female as she has 2 X chromosomes and 22
homologous (paired) chromosomes.









1


2



3




4



5











6


7


8 9




10


11 12









13 14 15 16 17 18













19 20 21 22 XX

If the above karyotype was taken from a male it would look identical except instead of chromosomes
it would h
ave one X and one Y chromosome as shown below.

X chromosome



Y chromosome


3.1.4

2

Describe one appl
ication of karyotyping (cross reference 3.3.4).


Karyotyping is used in pregnant women who are over 40 and are therefore at an increased risk of
having a baby with Downs Syndrome. Pre natal scanning or amnniocentesis occurs when a small
sample of amnioni
c fluid is removed by a long hollow needle from the pregnant mother’s amiotic fluid
which surrounds the baby and therefore contains some fetal cells. The fetal cells are cultured (grown in
the lab) and a karyotype made. If the fetus has Downs Syndrome th
ere is an extra chromosome in the
Karyotype (chromosome 21) As there are 3 chromosome 21’s the other name for Downs Syndrome is
Trisomy 21. Visit website
http://www.biology.arizona.edu/human_bio/activities/karyotyping/patient_a/patient_a.html

To complete and see the karyotype.


Downs Syndrome occurs due to the non
-
disjunction of chromosome 21


in other words during cell
division the homologous chromosom
es fail to separate. See 3.3.4 for more detail


Below follows afew genetic disorders


Diagnosis

Chromosomal Abnormality

Normal # of chromosomes patient's
problems are due to something other than an abnormal number of chromosomes.
Klinefelter's Syndrome one

or more extra sex chromosomes (i.e., XXY) Down's
Syndrome Trisomy 21, extra chromosome 21 Trisomy 13 Syndrome extra
chromosome 13



3.1.5

1

Define
gene
.

3.1.6

1

Define
allele
.

3.1.7


Define
genome
.


‘Gene’



a heritable factor that controls a specific
characteristic, consisting of a length of DNA
occupying a position on a chromosome known as a locus.




‘Allele’



one specific form of a gene, differing from other alleles by one or a few bases only and
occupying the same p
osition (locus) on the gene




‘Genome’
-

the total genetic material of an organelle, cell or organism




3.2

Gene mutation

(1h)

3.2.1

1

Define
gene mutation
.




Mu
tation is central to the theme 'Evolution'. Some
discussion could come in here about the overlap
between the frequency of the sickle cell allele and the
distribution of malaria. The terms 'point mutation' or
'frameshift mutation' will not be used.


‘Gene
mutation’


a change in the base sequence of a gene’ eg the gene reads ATC GAC CCC and
changes to ATC GCC CCC note how the bases have changed . Gene mutations occur spontaneously
in nature, but exposure to some chemicals and X
-
rays increases the chance o
f mutation. Mosyt
mutations are recessive, so are masked by the normal gene on the homologous chromosome. Most
mutations are not advantageous, but natural selection (survival of the fittest’ tends to eliminate harmful
mutations by causing death or infert
ility of the organism. Advantageous mutations increase the
survival of the individual eg


girraffes used to have short necks controlled by a gene for short necks


their offspring also had short necks. A mutation occurred in afew girraffes resulting in

a new long
necked variety. This had an advantage over the other girraffes as its longer neck meant in times of
food shortage it ate leaves from higher up the trees. The short necked gene died out as the short necked
giraffes failed to reproduce or died
due to lack of food. The mutated long neck gene was advantageous
and altered the gene pool of the species by wiping out the short neck gene.


In humans the gene for sickle cell anaemia is recessive and only causes the disease if both parents pass
on the re
cessive disease gene to the offspring, resulting in their death. If one disease gene and one
normal gene are passed on the offspring carries the disease but the effects are masked by the good
gene. In parts of Africa the sickle cell gene is common as it o
ffers some protection against Malaria.
The gene has not been wiped out as it offers heterozygous advantage to the carriers. Caucasions have a
lower % of sickle cell gene as malaria is not common in the UK and the gene has been almost wiped
out.


3.2.2

2

Outline the difference between an insertion and a deletion.




No mention of the causes of such events is required.



Insertion



The addition of new genetic material to a sequence

Eg


Old sequence


ATT

CGT

ATA

AAA

New sequence


ATT

T
CG

TAT

AAA

A


Only o
ne base has been added but has changed the whole triplet code resulting in new amino acids
being substituted and a new mutent protein made. In this case the insetion is a Thymine shown in bold.


Deletion


of a base involves the removal of genetic material

from a sequence

Eg




Old sequence


AT
T

CGT

ATA

AAA

New sequence


ATC

GTA

TAA

AA


In this case the underlined thymine has been deleted changing the whole triplet code resulting in new
amino acids being substituted and a new mutent protein made.


To see
the effects of insertions and deletions on the DNA, the RNA and the amino
acids substituted

the website below and click on link specified


http://library.thinkquest.org/3564/



DNA to Protein



3.2.3

3

Explain the consequence of a base substitution mutation in relation
to the process of transcription and translation, using the example of
sickle cell anaemia.


base substiotution
-

One of the bases is substituted making a new
amino acid for that codon, but unlike
insertions and deletions no knock on effect occurs with the otheer bases

Eg


Old sequence


A
T
T

CGT

ATA

AAA

New sequence


T
TT

CGT

ATA

AAA


In this example the adenine
A

has been substituted by thymine
T


In
sickle cell
anaemia

a base substitution occurs to one base out of 146 amino acids (438 bases) in the
gene for haemoglobin. This results in the normal amino acid, glutamic acid being substituted by the
amino acid, valine. The haemoghobin crystalises out in low oxygen

concentration causing the red
blood cells to change shape and block arteries which can be fatal. In the heterozygous case there is a
normal gene to mask this which is expressed.

GAA (codes for amino acid glutamic acid) base substitution of U instead of A
results in GUA (codes
for amino acid valine)




3.3

Meiosis

(2h)

3.3.1

1

State that meiosis is a reduction division in terms of diploid and
haploid numbers of chromosomes.


During sexual reproduction the

number of
chromosomes of an organism needs to be
halved so each sex cell has half of the genetic
material of the parent. As each chromosome
has a pair (homologous chromosome


see
karyotype) only one of each pair is passed onto
the egg or sperm. The tota
l number
of
chromosomes in the organism (diploid number)
is correct when the sperm and egg meet. The
type of cell division which halves the number
of chromosomes in the daughter cells (zygotes)
is called reduction division, and the daughter
cells are sai
d to have the haploid (half) number
of chromosomes.

In humans the diploid number is 46 and the
haploid number 23.










3.3.2

2

Outline the process of meiosis including pairing of chromosomes
followed by two divisions which result in four haploid cells.


1
st

cell division


A



Parent cell contains
chromosomes from mother
(red) and father (blue).
Chromosomes condense and
replicate.


B
-

Homologous (identical)
chromosomes pair up


C
-

Homologous chromosomes
line up on centre of cell




D
-

Homologous chr
omosomes
are pulled to poles (ends) of cell




E
-

Cell splits (cleaves) to make
2 daughter cells



2
nd

cell division


F
-

Chromosomes line up along
equator


G
-

Chromosomes migrate
towards poles


H
-

Cell cleaves to make 4
daughter cells with the haploid
nu
mber of chromosomes


Summary


In meiosis there are 2 divisions and the end result is 4 daughter cells with the haploid
(half) number of chromosomes.


3.3.3

3

Explain how the movement of chromosomes during meiosis can
give rise to genetic variety in the re
sulting haploid cells.

As shown in the resulting 4 daughter cells above they are not identical, some contain the long
chromosome from the mother, others the short from the mother, some contain the long chromosome
from the father, others the short chromos
ome. This results in the daughter cells all being different as
they could have different combinations of maternal and paternal chromosomes. This example only
uses 2 chromosomes from the mother and father, but as there are 46 chromosomes the amount of
dif
ferent information which could be in the daughter cell is huge. (the number of combinations is
worked out by
2
n
where n is the haploid number


in humans

2
23
=
8388608 different combinations)


Daughter cell

Paternal (fathers)
long chromosome

Maternal
(moth
ers) long
chromosome

Paternal (fathers)
short chromosome

Maternal
(mothers) short
chromosome

1

yes

no

yes

no

2

yes

no

no

yes

3

no

yes

yes

no

4

no

yes

no

yes


3.3.4

3

Explain that non
-
disjunction can lead to changes in chromosome
number, illustrated by

reference to Down's Syndrome (trisomy 21).



Left = normal meiotic division, right = non disjunction




In
normal meiotic division

one of the chromosomes (chromosome 21 see karyotype) is shown
dividing by meiosis. Normal
meiotic division of the parental chromosome 21 is shown on the left. The
parent cell has 2 identical (homologous) chromosomes, one from their mother and one from their
father. The daughter cells all have half the number of chromosomes (haploid number) so w
hen the sex
cell (gamete)

sperm
-

fuses with another sex cell
-
egg
-

there are 2 chromosome 21’s in the fertilised
egg (offspring.)


In Downs Syndrome
Non
-
Disjunction

occurs as shown in the left. Non Disjunction means the
chromosomes fail to separate during
the 1
st

meiotic division. Two of the resulting daughter cells are
empty, and 2 have the same amount of chromosomes as their parents (diploid number) When one of
the diploid daugher cells
-
sperm
-

fuses with the other sex cell
-
egg
-

the result will be a ferti
lised egg
with three copies of chromosome 21.(trisomy 21)


3.3.5

1

State Mendel's Law of Segregation.


Gregor Mendel was a monk in 1856 who carried out experiments on pea plants, and genetics.


He believed that the characteristics of an organism were dete
rmined by internal factors appearing in
pairs (we now call these chromosomes) and that only one a pair of factors could be present in a gamete
(sex cell)


3.3.6

3

Explain the relationship between Mendel's Law of Segregation and
meiosis.

Meiosis results in

one of each chromosome pair appearing in a gamete Meiosis results
in one of each chromosome pair appearing in a gamete which ties in with Mendel’s
Law above except Mendel used the word factors instead of chromosomes.