Topic 4.4

A crime scene(body tissue samples)

A single viral particle

(from an infection)

Using the technique called polymerase chain reaction (PCR), researchers are able to create vast quantities of DNA identical to trace samples. This process is also known as DNA amplification.

Many procedures in DNA technology

require substantial amounts of DNA to work with, for example;

DNA sequencing

DNA profiling/fingerprinting

Gene cloning

Transformation

Making artificial genesFragments of DNA from

a long extinct animal

Sometimes DNA samples can be hard to obtain:

The laboratory process called the polymerase chain reaction or PCR involves the following steps 1-3 each cycle:

Separate StrandsSeparate the target DNA strands by heating at 98°C for 5 minutes

Add Reaction MixAdd primers (short RNA strands that provide a starting sequence

for DNA replication), nucleotides (A, T, G and C) and

DNA polymerase enzyme.

IncubateCool to 60°C and incubate for a few minutes. During this time, primers

attach to single-stranded DNA. DNA polymerase synthesises complementary strands.

Repeat for about 25 cycles

Repeat cycle of heating and cooling until enough copies of the target DNA

have been produced.

Although only three cycles of replication are shown here, following cycles replicate DNA at an exponential rate and can make literally billions of copies in only a few hours.

The process of PCR is detailed in the following slide sequence of steps 1-5.

PCR

cycles

No. of target

DNA strands

1 2

2 4

3 8

4 16

5 32

6 64

7 128

8 256

9 512

10 1024

11 2048

12 4096

13 8192

14 16 384

15 32 768

16 65 536

17 131 072

18 262 144

19 524 288

20 1 048 576

21 2 097 152

22 4 194 304

23 8 388 608

24 16 777 216

25 33 554 432

Cycle 1

Cycle 2

Cycle 3

Original DNA Sample

Primer annealed

A DNA sample called the

target DNA is obtained

DNA is denatured (DNA strands

are separated) by heating the

sample for 5 minutes at 98C

Primers (short strands of mRNA)

are annealed (bonded) to the DNA

Nucleotides

Nucleotides

After one cycle, there are now two copies of the original sample.

The sample is cooled to 60°C.

A thermally stable DNA polymerase enzyme binds to the primers on each side of the exposed DNA strand.This enzyme synthesises a complementary strand of DNA using free nucleotides.

Media showcase animation

McGraw-Hill PCR animation

PCR song PCR song 2

PCR primers do not bind unless there is a complementary sequence of nucleotides◦ A = T◦ T = A◦ G ≡ C◦ G ≡ C

One test for GM ingredients in food involves a primer that will only bind to the GM DNA.

If GM DNA is present, the PCR process will amplify the DNA

If no GM DNA is present, the PCR has no effect

Gel electrophoresis can be used to separate large molecules (including nucleic acids or proteins) on the basis of their size, electric charge, and other physical properties.

DNA is split into fragments using restriction enzymes

The DNA samples are placed in wells and covered with a buffer solution that gradually dissolves them into solution.

Wells

Buffer solution

Cathode

AnodeGel

Plastic frame

Buffer

Sample

DNA has a negative charge because the phosphate groups are negatively charged.

The DNA fragments in the gel move through the gel towards the positive terminal of the electric field.

Smaller molecules move at a faster rate through the gel; longer fragments take longer to work through the small spaces in the gel.

Groups of DNA fragments can be seen as bands on the gel – usually seen with the help of a dye.

negative

terminal

positive terminal

Small fragments

Large fragments

Tray: Contains the set gel.

DNA solutions: Mixtures of different sizes of DNA fragments are loaded into each well.

DNA markers: A mixture of DNA molecules with known molecular weights. They are used to estimate the sizes of the DNA fragments in the sample lanes.

DNA fragments: The gel matrix acts as a sieve for the DNA molecules.

Wells: Holes created in the gel with a comb.

Gel electrophoresis demo lab

DNA profiling (DNA fingerprinting) is a technique for genetic analysis, which identifies the variations found in the DNA of every individual.

The profile refers to the distinctive pattern of fragments which is used to identify an individual.

DNA profiling does not determine a base sequence for a sample but merely sorts variations in base sequences.

Only one in a billion (i.e. a thousand million) persons is likely to have an identical DNA profile, making it a useful tool for forensic investigations and paternity analysis

the presence of a particular gene,(such as cystic fibrosis) in a family.

genetic relatedness of different organisms

e.g. checking on pedigree in stock breeding programs.

e.g. checking that captive populations of endangered species are not inbred.

DNA profiling can be used for investigating:

DNA fingerprints from tissue samples can be used as evidence in the same way traditional fingerprinting is used.

Which DNA fingerprint from the three suspects matches that of the tissue sample submitted as evidence?

Why would the DNA from the victim be included in this test?

The DNA from the victim must be excluded from the evidence.

In a paternity test, the DNA from the mother must also be included to exclude her contribution to the banding patterns in the child’s profile.

DNA profiling can be used for forensic purposes:

We expect 100% match as the cells left behind at the scene are the perpetrator’s cells

We expect 100% match as the cells left behind at the scene are the perpetrator’s cellsThe overlapping bands between victim and suspect indicate a close genetic relationship

No. Without a stronger match, the evidence is insufficient to convict the suspect. He should be released and a new suspect found.DNA evidence is being reviewed in many wrongful convictions.

Because the child inherits half its genetic material from each parent, any band that the child has not inherited from his mother, he must have inherited from his father.

In this case, determine which man (1 or 2) is the biological father of the child.

1. Refer to the image on the rightWhich male (1 or 2) is the father of the child?Explain.

2. Refer to the image on the leftWhich suspect(1,2 or 3) was present at the crime scene?Explain.

“DNA is better at proving innocence than guilt.”

Discuss this statement

Textbook exercises:

Tiger book p164Look at figure 7 and determine the culprit

Tiger book p 166Answer question 1 on logarithmic scales in Biology

Tiger book p167Question 6

4.4.3 4.4.3 Outline three outcomes of the Outline three outcomes of the sequencing of the complete sequencing of the complete

human genomehuman genomeCompleted in April 2003.An international, collaborative effort to record the entire base sequence of the human genome.Also achieved the following:

Discovered the number and loci of all the genes (30k) in our genome – further research in diagnostics, treatment and pharmacology.New proteins and their functions were discovered.Comparisons between genomes of different species – evolutionary history and links.Bioinformatics was born – a high-tech way to collate and access information from genetic databases.

Discussing the implications of the HGP

Scrubs – Huntington’s Disease1. If you knew that a member of your family had a rare genetic disorder

and you could be tested for it quickly and easily, would you do it? Why?

Human Genome Project - Ethical, Legal, & Social Implications2. If you were invited to share your genome with researchers in the

hope of finding cures for genetically-based illnesses, would you do it? Why?

Robert Cook-Deegan on Patenting Genes3. Do you feel that gene patenting should be allowed? Why?

4.4.7 When genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code

is universalAll living things use the same bases (G.A.T.C!)A particular codon will produce the same amino acid, regardless of the species.This means the sequence of amino acids in a polypeptide remains unchanged.This allows us to take a gene from one species and insert it into the genome of another species.A well-known example of this gene transfer process is the production of human insulin by GM E. coli bacteria.A potential treatment for haemophilia is the injection of human clotting factors produced in the milk of GM sheep.

4.4.8 A basic technique for gene transfer involves plasmids, a host cell, restriction enzymes and DNA

ligase.

Recognition SiteRecognition Site

GAATTC

CTTAAG

DN

A CTTAAG

GAATTC

cutThe restriction

enzyme EcoRI cuts

here

cut cut

Naturally occurring bacterial enzymes are used as “molecular scalpels”

Allow genetic engineers to cut up DNA in a controlled way.

Restriction enzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites.

It is possible to use restriction enzymes that cut leaving an overhang; a so-called “sticky end”.

DNA cut in such a way produces ends which may only be joined to other sticky ends with a complementary base sequence.

See steps 1-3 opposite:

C T T A A

A A T T C G

G

FragmentRestriction

enzyme: EcoRI

Sticky endRestriction enzyme: EcoRI

DNA from

another

source

A restriction enzyme cuts the double-

stranded DNA molecule at its specific

recognition site

The two different fragments cut

by the same restriction

enzyme have identical sticky

ends and are able to join

together

The cuts produce a

DNA fragment with

two “sticky” ends

When two fragments of DNA cut by the same

restriction enzyme come together, they can join by

base-pairing

C T T A A

A A T T C

G

G A A T T C

C T T A AG

G

C T T A A

A A T T C G

G

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

It is possible to use restriction enzymes that cut leaving no overhang; a so-called “blunt end”.

DNA cut in such a way is able to be joined to any other blunt end fragment, but tends to be non-specific because there are no sticky ends as recognition sites.

Restriction enzyme

cuts here

Recognition Site Recognition Site

DNA from another source

The cut by this type of restriction

enzyme leaves no overhang

cutcut

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

G G G

G G G

C C C

C C C

DNA

A special group of

enzymes can join

the pieces together

DNA fragments produced using restriction enzymes may be reassembled by a process called ligation.

Pieces of DNA are joined together using an enzyme called DNA ligase.

DNA of different origins produced in this way is called recombinant DNA because it is DNA that has been recombined from different sources.

Steps 1-3 are involved in creating a recombinant DNA plasmid:

Plasmid DNA fragment

Two pieces of DNA are cut using the same restriction enzyme.

Foreign DNA fragment

A A T T C G

C T T A AG

The two different DNA fragments are attracted to each other by weak hydrogen bonds.

This other end of the foreign DNA is attracted to the

remaining sticky end of the plasmid.

When the two matching “sticky ends” come together, they join by base pairing. This process is called annealing.

This can allow DNA fragments from a different source, perhaps a plasmid, to be joined to the DNA fragment.

The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid.

Detail of Restriction Site

Restriction sites on the fragments are attracted by base pairing only

Gap in DNA molecule’s ‘backbone’

Foreign

DNA

fragment

A A T TC

A A T T C

G

G

C A

Plasmid

DNA

fragment

G

G

T T A

AATTC

DNA ligase

The fragments are able

to join together under the

influence of DNA ligase.

GA A T T

C

G A A T T C

C T T A A G

GAATT

C

Recombinant Plasmid DNA

Detail of Restriction Site

Fragments linked

permanently by

DNA ligase

No break in

DNA molecule

The fragments of DNA are joined together by the enzyme DNA ligase, producing a molecule of recombinant DNA.

These combined techniques of using restriction enzymes and ligation are the basic tools of genetic engineering.

A virus vector is used to insert the recombinant plasmid into the genes of affected cells.

The virus is chosen or designed to target only those specific cells.

Severe Combined Immune Deficiency can be treated this way

Gene therapy ‘reverses’ hereditary blindness

Plant example

s

Golden rice: Enriched with beta-carotene, which is converted to Vitamin A in the body. Can prevent malnutrition-related blindness in developing countries.

Insect-resistant corn: Produces proteins which pests do not like, therefore toxic pesticides are not needed on the farm.

Salt-resistant tomatoes: Can be grown in soil with a high saline concentration.

Animal example

s

Factor IX-producing sheep: Produce human clotting factors in their milk , for the treatment of haemophilia.

Glowing pigs: Cells from these organisms are used to study transplants and grafts, and the final destinations of transplanted cells in the host body.

Enviropig: Produce phytase in their saliva to convert insoluble phytate into phosphate that is absorbed by the pig.

4.4.9 Give examples of the current uses of genetically modified crops or animals

GMOs are already in circulation and have been produced for many uses, including agricultural and medical.

The ethical debate over GMOs rages on, and as scientists we must always bear in mind the precautionary principle:

“If an action is potentially harmful, the burden of proof of safety lies with those who propose to take the action.”

Benefits

Increased yields of crops and faster breeding cyclesCrops can be grown in harsher environmental conditionsReduced need for pesticides which can harm human and environmental health through biomagnificationNutrient-enriched crops in areas of high food pressures or famine

Potential harms

Potential genetic pollution of organic crops through fertilisation by pollen of GM cropsUnknown health risks of some cropsFear of monopoly-like behaviour as farmers need to buy expensive seeds annuallyPotential hybridisation of related species

GM food and you

4.4.10 Discuss the potential benefits and possible harmful effects of one example of

GM

A group of genetically identical organisms or groups of cells derived from a single parent cell

Monozygotic (one zygote) twins are naturally occuring clones.

Why do they not look identical?... Epigenetics has the answer

Asexual reproduction (i.e. in bacteria) is an example of cloning

Cloning via binary fission

Taking plant cuttings and growing a new plant is also cloning.

As is plants producing bulbs and runners

4.4.11 Define clone

Cloning cell cultures using nuclear transfer:

1. Remove a differentiated diploid nucleus from the individual to be cloned

2. Enucleate a donor egg cell3. Insert the diploid nucleus into the enucleated

egg cell4. Stimulate it to divide and grow5. Collect cells for therapeutic purposes, such as

creating skin tissue for burn patients

4.4.12 Outline a technique for cloning using differentiated animal cells

Reproductive cloning using nuclear transfer:1. Remove the differentiated diploid nucleus

from the individual to be cloned2. Enucleate the donor egg cell3. Insert the diploid nucleus into the enucleated

egg cell4. Insert into a surrogate mother and gestate5. The newborn will be genetically identical to

the donor nucleus parent

Reproductive cloning

The first mammal cloned was Dolly the sheep

Human cloning is illegal around the world

There will be no: “Mini-me” as in ‘Austin Powers’ (a clone to take over the world) or the characters played by Ewen McGregor and Scarlett Johansen in ‘The Island’ (spare parts “just in case”).

Therapeutic cloning is less controversial

It has the potential to treat (using stem cells) degenerative diseases like Parkinson’s disease and Multiple Sclerosis.

Does involve producing an embryo that could grow to full term (same as an IVF embryo) – but then it would be cloning!

Dolly and her birth mom.

A black faced ewe cannot have a white faced baby – so they

must not be genetically related.

In 2008, a team led by DR Viviane Tabar extracted skin cells from the tails of mice with Parkinson’s disease.

They removed the nucleus from each cell and implanted them into egg cells from which the nuclei had been removed.

The resulting cells, which were genetically identical to the donor mice, developed as embryos and produced stem cells that could differentiate into dopamine neurons – the type that are missing in Parkinson’s disease.

The team injected these stem cells into the affected regions of the brains of the donor mice and found that there was a marked improvement in the symptoms of Parkinson’s disease.

Is it acceptable to create embryos as a source of stem cells for treatments

that reduce suffering in a child or adult, but result in the death of the

embryo?

Nucleus is removed

Nucleus is removed

Possible benefits Arguments against

Rejection risk reduced in transplants

Religious objections to “playing God” by creating what many

consider to be human life

No need to wait for human donor to die to give organs UN recommendations against

reproductive cloning has no been ratified by all countries – possible risk of a race to create

the first human cloneSome success stories already reported in therapeutic cloning

4.4.13 Discuss the ethical issues of therapeutic cloning in humans