Applications of Applications of DNA technologyDNA technology

Human Genome ProjectHuman Genome ProjectHuman TherapeuticsHuman Therapeutics

Forensic UsesForensic UsesAgricultureAgriculture

HistoryHistory

Late 1980’s idea was proposedLate 1980’s idea was proposed Predicted it would take 15 yearsPredicted it would take 15 years Cost about $200 million per yearCost about $200 million per year

$1 per base pair$1 per base pair Officially began in 1990Officially began in 1990 26 June 2000 joint announcement from Blair 26 June 2000 joint announcement from Blair

and Clinton ‘the draft complete’and Clinton ‘the draft complete’ Joint publication in Nature and Science 12 Joint publication in Nature and Science 12

Feb 2001Feb 2001 14 Apr 2003 – The finished human genome14 Apr 2003 – The finished human genome

Why?Why? ‘‘If I were to study the carburettor of my If I were to study the carburettor of my

car engine, even in exquisite detail, I car engine, even in exquisite detail, I would still have no idea about the overall would still have no idea about the overall function of the engine, much less the function of the engine, much less the entire car. To understand what an entire car. To understand what an engine is for, and how it works, I’d need engine is for, and how it works, I’d need to study the whole thing – I’d need to to study the whole thing – I’d need to place the carburettor in context, as one place the carburettor in context, as one function part among many. The same is function part among many. The same is true of genes…..’true of genes…..’

James WatsonJames Watson

MethodMethod

Genetic MappingGenetic Mapping Identifies relative positions of genesIdentifies relative positions of genes E.g. Gene 2 lies between genes 1 and 3E.g. Gene 2 lies between genes 1 and 3

Physical MappingPhysical Mapping Absolute positions of genes on Absolute positions of genes on

chromosomeschromosomes E.g. Gene 2 is 1 million bp from gene 1E.g. Gene 2 is 1 million bp from gene 1

DNA sequencingDNA sequencing Actual ATCG combinationsActual ATCG combinations

Polymerase Chain Polymerase Chain ReactionReaction

Critical to the HGP was the ability to create Critical to the HGP was the ability to create large amounts of DNA for sequencing.large amounts of DNA for sequencing.

PCR is a process to amplify DNAPCR is a process to amplify DNA DNA is heated to 95oCDNA is heated to 95oC The DNA is denatured causing the two strands The DNA is denatured causing the two strands

to separateto separate A primer (short length of DNA) binds (or A primer (short length of DNA) binds (or

anneals) to the template strands [after the anneals) to the template strands [after the solution is cooled]solution is cooled]

Complementary DNA strands form [through the Complementary DNA strands form [through the action of DNA polymerase]action of DNA polymerase]

Genetic MappingGenetic Mapping

Genetic mapping utilises cross over Genetic mapping utilises cross over frequencies between known genetic frequencies between known genetic markers.markers.

A genetic marker is any sequence of A genetic marker is any sequence of genome that shows difference genome that shows difference between individuals. It could bebetween individuals. It could be A geneA gene A Microsatellite or Short Tandem A Microsatellite or Short Tandem

RepeatRepeat

Microsatellites (STRs)Microsatellites (STRs)

Repeating sequences 2 – 4 nucleotide bases Repeating sequences 2 – 4 nucleotide bases found in mostly in introns (‘junk’ DNA)found in mostly in introns (‘junk’ DNA)

The number of repeats varies from person The number of repeats varies from person to person, but follow patterns of Mendalian to person, but follow patterns of Mendalian inheritanceinheritance

These sequences can be identified using These sequences can be identified using probes. A probe isprobes. A probe is A short, single stranded sequence of DNAA short, single stranded sequence of DNA Complementary to DNA base sequenceComplementary to DNA base sequence ‘‘Tagged’ – radioactively or using fluorescent dyeTagged’ – radioactively or using fluorescent dye

Recombinance / Cross Over Recombinance / Cross Over FrequencyFrequency

What do you remember about linked What do you remember about linked genes from Higher.genes from Higher.

What information do recombinant What information do recombinant individuals give us about the relative individuals give us about the relative position of genes?position of genes?

Physical MappingPhysical Mapping

Use of restriction enzymesUse of restriction enzymes Restriction enzymes belong to the group Restriction enzymes belong to the group

‘endonucleases’ or nucleases.‘endonucleases’ or nucleases. They cut DNA at specific sequences.They cut DNA at specific sequences. Chromosomes can therefore be cut into Chromosomes can therefore be cut into

sequences of different lengths.sequences of different lengths. By using combinations of restriction By using combinations of restriction

enzymes and working out the size of the enzymes and working out the size of the fragments, a pattern of recognition sites fragments, a pattern of recognition sites in the DNA can be pieced together.in the DNA can be pieced together.

Gel electrophoresisGel electrophoresis Uses physical properties of DNA (size and Uses physical properties of DNA (size and

charge) to separate moleculescharge) to separate molecules Gel electrophoresis involves running an Gel electrophoresis involves running an

electric current through an agarose gel. DNA electric current through an agarose gel. DNA is loaded into wells at the negative end.is loaded into wells at the negative end.

DNA is repelled, and moves through the DNA is repelled, and moves through the agarose gel at different speeds depending on agarose gel at different speeds depending on the size of a fragment.the size of a fragment.

A Molecular weight marker (of known A Molecular weight marker (of known fragment size) is also used. Samples can be fragment size) is also used. Samples can be compared to this marker to work out their compared to this marker to work out their size.size.

Physical Mapping cont…Physical Mapping cont…

M = molecular weight M = molecular weight markermarker

1 = undigested 1 = undigested samplesample

2 = digested with NotI2 = digested with NotI

3 = digested with 3 = digested with BamHIBamHI

4 = digested with NotI 4 = digested with NotI and BamHIand BamHI

5 = something totally 5 = something totally differentdifferent

Physical Mapping cont…Physical Mapping cont…

Physical Mapping cont….Physical Mapping cont….

DNA SequencingDNA Sequencing

Sanger method (aka dideoxy chain-Sanger method (aka dideoxy chain-termination method)termination method) An unknown DNA template strand is An unknown DNA template strand is

replicated using a primer (to initiate replicated using a primer (to initiate replication), DNA polymerase, nucleotide replication), DNA polymerase, nucleotide bases and one of 4 dideoxy nucleotide bases and one of 4 dideoxy nucleotide bases (ddA, ddG, ddC and ddT)bases (ddA, ddG, ddC and ddT)

DNA sequencing cont…DNA sequencing cont… If a ddNTP is inserted instead of a If a ddNTP is inserted instead of a

normal nucleotide DNA replication normal nucleotide DNA replication stops at that point (chain termination) stops at that point (chain termination)

When this experiment is repeated with When this experiment is repeated with other dNTPs DNA molecules differing other dNTPs DNA molecules differing in length by 1 bp are created.in length by 1 bp are created.

DNA sequencing cont..DNA sequencing cont.. These are then run on an electrophoresis gelThese are then run on an electrophoresis gel

Note: The smallest pieces at the start of the Note: The smallest pieces at the start of the sequence are at the bottom. Therefore the sequence are at the bottom. Therefore the sequence is read bottom to topsequence is read bottom to top

DNA sequencing cont…DNA sequencing cont…

Comparing GenomesComparing Genomes

Other genomes have been sequenced in Other genomes have been sequenced in conjunction with the human genome, and conjunction with the human genome, and have continued.have continued.

There are a large number of similarities There are a large number of similarities between genomes.between genomes.

Genes found in a wide range of organisms Genes found in a wide range of organisms are known as homologous genes.are known as homologous genes.

These are used to study function of These are used to study function of genes.genes.

Learning ActivitiesLearning Activities

Look at the arrangements document Look at the arrangements document to clarify what information is to clarify what information is required.required.

Read DART pg 73 – 81.Read DART pg 73 – 81. Read the Monograph pg 67 – 79Read the Monograph pg 67 – 79 Scholar – 8Scholar – 8 Internet researchInternet research WorksheetsWorksheets