Unit 1Cell and Molecular

Biology

Section 9

Human therapeutics and forensic uses

What is DNA?

DNA is the carrier of genetic information and provides a structural plan for proteins.

It consists of linear linked nucleotides whose sequence forms hereditary.

The DNA is in the form of a double helix and is made up of four bases: adenine, thymine, cytosine and guanine.

DNA Uses for Forensic Identification

DNA is used in forensics to: Identify potential suspects if their DNA matches DNA found at

crime scene Prove possible innocence of people wrongly accused of crime Identify crime and catastrophe victims Show paternity and other family relationships Identify endangered/protected species (can be used to prosecute

poachers) Detect bacteria polluting air, soil, water, food Match organ donors with organ receivers Determine pedigree

Solving Crimes

DNA can be used to identify criminals with incredible accuracy when biological evidence exists.

Still not used to convict people for a long time as juries didn’t understand how the DNA evidence proved anything.

Samples could be contaminated easily.

DNA Fingerprinting

This works on the principle that individuals poccess regions within their chromosomes that have short repeat sequences

The number of times a sequence repeats is unique to each individual

These regions are known as minisatellite regions with the repeats being known as variable number tandem repeats VNTR

In forensics specific enzymes are used to cut the DNA at these regions and the fragments separated by electrophoresis

Since every person has a unique number and pattern of VNTR they will produce a DNA fragments which vary in number and size – this shows up as a unique banding pattern

Who is the Daddy?

In this example a single restriction enzyme has cut the DNA at specific points generating a small number of fragments.

DNAprofi ling:Single-locusprobe

M 1 2 3 4 F

maternalbands

paternalbands

children

Who is the Baddy?

In this example a variety of restriction enzymes have cut the DNA at many specific points generating a large number of fragments.

DNAprofiling:Multi-locusprobe

Southern Blot Technique

DNA fingerprinting uses a technique known as southern hybridisation and also involves genetic probes to target stable areas on either side of the minisatellite regions. There are six main steps

1. DNA extraction and amplification of sample

2. Digestion of DNA with a restriction endonuclease

3. Agarose gel electrophoresis

4. Preparation of a "southern blot" DNA fragments are transferred to the surface of a nylon membrane by blotting. This denaturation/blotting procedure is known as a "Southern Blot" .

The blotting of DNA to a nylon membrane preserves the spatial arrangement of the DNA fragments that existed after electrophoresis.

5. The southern blot is hybridised with a radioactive probe. Probes are radioactive sections of DNA or RNA with a base sequence complementary to the region being targeted. In this case a single locus on one chromosome

6. Autoradiography - Pressing the southern blot onto photographic paper shows the regions bound to the probe as black lines or blots.

Cystic Fibrosis (CF)The CF gene codes for a membrane carrier protein present in epithelial cells called CF transmembrane conductance regulator (CFTR). This controls the passage of chloride ions across the cell membrane.

In patients with cystic fibrosis the structure of the protein is altered resulting in problems with the flow of chloride and sodium ions into and out of the cell across the membrane.

The lung is the main organ affected by CF. If the chloride channel is blocked the airway becomes drier. This causes sticky mucus to build up in the lungs.

· Major symptoms: inflammation of lung tissue and persistent bacterial infection.

· Other symptoms: defects in the pancreas.

The disease is relatively easy to diagnose as a symptom is the production of salty sweat.

Affected individuals have a reduced quality of life and a life expectancy of about 30 years.

Daily physiotherapy and drugs are used to rid the lungs of the build up of mucus.

Diseases caused by a single-gene defect are known as monogenic traits and are characterised as either:1. Autosomal dominant2. Autosomal recessive3. X-linked (sex-linked) which are mostly recessive

Diseases that involve several genes are known as polygenic traits and they are usually more difficult to diagnose and treat than the single-gene defects.

Cysti fibrosis – case study

CF shows a simple Mendelian inheritance pattern as it is a an autosomal recessive monogenic

trait (mutation).

Cystic fibrosis is caused by a single faulty gene NORMAL = Cf ABNORMAL = CfWe each carry TWO alleles for the Cf gene, thus:

NORMAL = Cf Cf CARRIER = Cf Cf AFFECTED = Cf Cf

Cystic fibrosis appears when two carriers produce a child:

Cf Cf

Mother

Cf

Cf

Father

Cf Cf Cf Cf

ch ro m o so m e 7b an d7q22

C F

0 100 200 300 400 500 kb p

280 kb con tig

? 1 2 3 4

C F T R gen e

p hy sical m arkers

The gene linked to CF was identified on chromosome 7.

There are over 700 different mutations that have been identified in the CFTR gene which helps, in part, to explain why different CF patients are affected to different degrees.

The variation in how the disease is expressed in the phenotype may also be due to environmental factors and the effect of modulator genes on the CFTR gene.

During genetic screening 12 of the mutations are currently tested for, one of which is ΔF508. Δ indicates that it is a deletion mutation, F is phenylalanine and 508 is the position of the deletion in the protein. The ΔF508 mutation accounts for 70% of CF cases worldwide.

(see the following two slides)

The cystic fibrosis F508 mutation (1)

Diagram how transcription produces the primary RNA transcript. This is converted into the functional mRNA by removal of intervening sequences.

On translation the CF transmembrane conductance regulator protein (CFTR) is produced.

Diagram shows the normal and mutated proteins. Normal CFTR has phenylalanine (F) at position 508. In the mutant form this is deleted, causing the protein

to fold incorrectly.

The cystic fibrosis DF508 mutation (2)

Test for cystic fibrosis defective allele

A short sequence that spans the mutated region is amplified using the polymerase chain reaction.

In lane 1 a normal homozygous pattern is shown. In lane 2 a carrier (hererozygous) shows two bands, one normal and one

smaller by 3 nucleotides (one codon, representing the deleted phenylalanine).

In lane 3 a CF-affected individual (homozygous recessive) shows one band at the lower position.

Duchenne’s Muscular Dystrophy (DMD) Duchenne’s muscular dystrophy is a X-linked disease that

affects around 1 in 3,300 boys.

It causes progressive wasting of the muscles so that by the time the person is in their teens they are likely to be confined to a wheelchair.

The gene was identified in 1987. The gene codes for the protein dystrophin.

Dystrophin’s normal function is to link the cytoskeleton with the sarcolemma (muscle cell membrane) in muscle cells.

Life expectancy is similar to CF, i.e. about 30 years.

Human Therapeutics

The techniques discussed in the previous lessons are now used to detect genetic disorders such as cystic fibrosis and Duchenne’s muscular dystrophy. This can lead to the development of a screening test which must be used in conjunction with counselling.

Congenital abnormalities are genetically-based diseases (often simply called genetic diseases) that are present at birth.

Cystic fibrosis and Duchenne’s muscular dystrophy are two examples of genetic diseases where molecular genetics has led to improved diagnosis and treatment.

Genetic diseases are first recognised by the disease symptoms and there are a number of steps required to establish the definitive genetic cause:

1.Trace the disease through family relationships by carrying out pedigree analysis to determine if the faulty gene is dominant, recessive or X-linked.

2.Once the disease has been identified as a monogenic trait then a search for the gene defect can take place using the processes similar to those employed for the human genome project, i.e.

Genetic mapping – by looking for genetic markers that are co-inherited with the disease. The more often the faulty gene and marker are co-inherited, the closer they are on the chromosome.

Physical mapping DNA sequencing

Cystic fibrosis: pedigree analysis

CF shows a simple Mendelian inheritance pattern as it is a an autosomal recessive monogenic

trait (mutation).

Cystic fibrosis is caused by a single faulty gene NORMAL = Cf ABNORMAL = Cf

We each carry TWO alleles for the Cf gene, thus: NORMAL = Cf Cf CARRIER = Cf Cf AFFECTED = Cf Cf

Cystic fibrosis appears when two carriers produce a child:

Cf Cf

Mother

Cf

Cf

Father

Cf Cf

Cf Cf Cf Cf

Cf Cf

Searching for the mutated gene

The gene was eventually identified in 1989 by examining cloned DNA fragments using the techniques of chromosome walking and chromosome jumping shown on the following two slides

Often a mixture of ‘walks’ and ‘jumps’ is needed to progress as shown in the diagram above.

Chromosome walking

In chromosome walking the end of each overlapping fragment is used in a hybridisation test to identify the next fragment.This is often used to ‘walk’ from a marker gene towards the target gene.

Chromosome walking

Chromosome jumping is a similar technique to chromosome walking but in this case a special cloning technique is used to isolate complementary fragments that are far apart.

This enables a ‘jump’ along the chromosome which is useful if the marker gene is far from the target gene.

Using the three techniques of genetic mapping, physical mapping, genetic mapping and DNA sequencing have allowed the position and sequence of the mutation to be identified.

Once this information is known it is possible to produce a tests which can identify the mutation if present in an individual. This involves the use of hybridised DNA and gene probes

Steps DNA sample taken from patient, cut with restriction

enzymes and run through electrophoresis gel The gel is blotted using the Southern Blot The DNA is hybridised with a radioactive probe – this

binds to the mutated gene Autoradiography shows the presence of the mutated

gene

Testing for the CF allele

N o rm al gen e sequ en ce 5' - G A A A A T A T C A T T G G T G T T T C C - 3'C T T I le I le P h e G ly

M utan t gen e sequ en ce 5' - G A A A A T A T C A T T G G T G T T T C C - 3' I le I le G ly

The 3-bp deletion:

Amplify this region using PCR:

Use primers about 100 bp apart

Normal allele produces 100 bp fragment, mutant a 97 bp fragment

A PCR-based test is used to identify the cystic fibrosis (CF) defective allele. After amplifying a short DNA fragment which spans the area of the deletion, the DNA fragments are then separated on an electrophoresis gel. (see the following slide)

NOTE: In lanes 1 and 3, shown on the following slide, the DNA band contains sequences from both paternal and maternal chromosomes that run to the same position in the gel. It is only in the heterozygous case that the two bands are distinguished.

Test for cystic fibrosis defective allele

A short sequence that spans the mutated region is amplified using the polymerase chain reaction.

In lane 1 a normal homozygous pattern is shown. In lane 2 a carrier (hererozygous) shows two bands, one

normal and one smaller by 3 nucleotides (one codon, representing the deleted phenylalanine).

In lane 3 a CF-affected individual (homozygous recessive) shows one band at the lower position.

Gene therapy for cystic fibrosis

( a ) I n v i vo gen e th erapy

( b ) E x v i vo gen e th erapy

in sert tran sgen ein to lip o so m es o rlip o p lex es, o r v iralv ecto r/ v eh ic le system s

g ro w cells ex vi vo

rem o v e cells

rep lace th em o d ifi ed cells

ad d tran sgen e an dselect m o d ifi ed cells

d eliv er to site o factio n ( lu n g) b y

aero so l sp raye.g.

Human therapeutics – Gene therapy

1. The possibility of replacing a defective gene with a ‘good’ copy of the gene to overcome the problems caused by the defect is called gene therapy. In this way one is able to deal with the cause not just the symptoms.

2. Gene therapy might also be used to kill abnormal cells, such as cancerous cells, or to inhibit the spread of viruses by preventing DNA replication.

3. In the case of cancer, gene therapy would avoid many of the side-effects associated with the drugs used in chemotherapy.

4. The difficulty with gene therapy in practical terms is that there are difficult criteria to be met before it can be used. The conditions are:

• the defective gene function is characterised;• the normal gene is available in cloned form;• the affected cells are accessible, either the

cells can be treated outside the body (ex vivo) and replaced, or may have to be treated within the patient’s body (in vivo);

• there are suitable vehicles (vectors) for delivery of the gene e.g. viruses, liposomes or artificial chromosomes

viral vectors – virus have evolved to alter a host cells metabolism by implanting their DNA into the host cells DNA. Altered viruses with therapeutic genes can be given to patient with virus implanting therapeutic gene. ( risky)

the use of liposomes that can fuse with cell membranes

human artificial chromosomes - the gene functions normally in its target cells.

The difficulty of meeting the criteria means that gene therapy is still in the early stages of development although progress is being made.

Gene therapy which raises ethical questions in the type of cells that are the targets, i.e. whether somatic cells (body cells) or germ cells are used. If genes within reproductive cells could be altered then the alteration could be passed on to the next generation and effectively alter the species gene pool.