Applications of DNA Technology:

All references made are to the unit 1 Scholar book. Page numbers may vary each year as new versions of the book are released.

• begun in 1990, started up by James Watson

• aim was to map the entire human genome – locate 20-25,000 genes and determine their function

• also to determine the base sequence for each gene.

• draft released 2000, completed 2003

• data still being analysed to understand what all genes do, and to see if the information will help in medical research

In this section, you are going to learn the techniques used in the human genome project to map out the human genome:

1.Genetic mapping

2.Physical mapping

3.DNA sequencing

4.PCR

However, back to basics first! (For Higher Biologists anyway!)

Monohybrid Inheritance

•One copy of each gene from each parent

•Each gene has different alleles

•Alleles present in genotype affect the phenotype

•Assume we can all remember how to do a punnet square to do a genetic cross?!

Dihybrid Inheritance

•A genetic cross involving 2 genes

•E.g. Gene A/a and B/b

P AABB x aabb

Chromosomes

Gametes AB ab

F1 AaBb

A B

A B

a b

a b

A B

a b

Now do a cross of the F1

P AaBb x AaBb

Gametes AB, aB, Ab, ab (for both parents)

AB aB Ab ab

AB AABB AaBB AABb AaBb

aB AaBB aaBB AaBb aaBb

Ab AABb AaBb AAbb Aabb

ab AaBb aaBb Aabb aabb

Expected ratio is 9:3:3:1

9 = Dominant for both A and B

3 = Dominant A, recessive b

3 = Recessive a, dominant B

1 = Recessive a, recessive b

P AaBb x aabb

Remember the chromosomes.

What are the possible gamete combinations for each parent?

Do a cross between the 2 parents to work out the genotypes for the offspring.

Offspring: AaBb, Aabb, aaBb, aabb

AB, Ab, aB, ab and ab

For the heterozygous parent, where does the aB and Ab combinations come from?

Their original chromosomes:

A B

a b

Crossing over at meiosis!

AB and ab are the parental genotypes.

Ab and aB are the new combinations produced by crossing over.

This is called recombination.

Offspring possessing the new combinations are called recombinants.

Genetic Mapping

•Linked genes – 2 genes carried on same chromosome

•Linked genes become split during crossing over

•Genes further apart will have a higher frequency of recombination

•Frequency of recombination (or crossover value) can be worked out by following formula:

Number of F2 recombinants

Total F2 offspringX 100

•The % of recombinants = map units

•Map units give an indication of distance between genes

•Used to plot a genetic map of a chromosome

•e.g. 12% recombinants for genes A and B would be represented by:

A B12

Try the mapping questions in scholar.

p.150 Q16 + p.151 Q17

Physical Mapping

•Genetic marking gives an indication of the distance between genes on a chromosome

•Physical mapping gives you the ‘true’ distance between genes in number of base pairs

•DNA is cut into fragments by restriction endonucleases

•Restriction endonucleases cut DNA at specific base sequences

•Using 1 or a combination of these enzymes on the same DNA strand gives you many fragments with overlapping sequences

•By comparing overlapping sequences, fragments can be put in order as they would appear on the original DNA strand

•Restriction endonucleases are found in bacteria (used as a defence mechanism to destroy invading pathogen’s DNA)•DNA fragments are separated according to size by gel electrophoresis http://www.dnalc.org/resources/animations/gelelectrophoresis.html

•Negatively charged DNA moves through gel to positive electrode.

• smaller fragments can move through the gel further than larger fragments

Try Q12 from 2009 Past Paper (Your answer may be the reverse of what is there, that’s allowed!) C

8 2

7 3

E B

7 1 2

Not an option!

E B

8 2

73

3 5 2

This is the reversed answer of C! Turn them the other way, and you’ll get C as it is in the paper!

DNA Sequencing

Based on ‘dideoxy chain termination method’ developed by Fred Sanger in the ’70’s

1.Make copies of your DNA fragment (see PCR later)

2.Add DNA, DNA polymerase, and a radioactive primer to 4 containers

Primer – short strand of nucleotides which mark the portion to be sequenced

3. In container 1 add ddATP, 2 – ddTTP, 3 – ddCTP, and 4 – ddGTP

‘dd’ nucleotides – dideoxyribonucleotides, when incorporated into the growing DNA strand, they terminate sequencing

4. In each reaction vessel new strands are synthesised – starts at primer, stops at ddnucleotide

5. Strands separated by gel electrophoresis

6. X-ray photo, or fluorescent labels show the electrophoresis results.

7. Sequence from strands can be read, from that, can then work out the original template.

Bead activity

Polymerase Chain Reaction (PCR) (Figure 8.3 p.145)

Used to produce many copies of a particular DNA sequence

Stages:

1.Heat to ~95oC to denature DNA (make single strands)

2.Cool so primers can bind to DNA

3.Taq polymerase extends 3’ end of each primer to make new strands

4. Repeat cycle to make more strands!

Comparative Genome Analysis

•Genomes of other species sequenced whilst sequencing human genome

•Allows genes homologous to humans to be mapped, to help determine their function.

•Size and complexity increases as complexity of organism increases

•Some anomalies – some plants have larger genomes than humans.

Nematode Worms (1996)

•33% similar to humans

•Used by researchers to carry out drug testing

Fruit Fly (2000)

•60% of 289 disease genes in humans, have equivalents in flies

•50% fly proteins similar to mammalian proteins

Mouse (2002)

•More than 90% of mouse proteins show similarities to known mammalian proteins

•New drugs regularly tested on mice – very close to human genome