Molecular Molecular Genetic Genetic

TechnologiesTechnologiesGel ElectrophoresisGel Electrophoresis

PCRPCRRestriction & ligation EnzymesRestriction & ligation Enzymes

Recombinant plasmids and Recombinant plasmids and transformationtransformation

DNA microarraysDNA microarraysDNA profilingDNA profiling

Gel ElectrophoresisGel Electrophoresis DNA is negatively chargedDNA is negatively charged Uses an agarose gel which contains Uses an agarose gel which contains

tiny wells in which DNA samples can be tiny wells in which DNA samples can be placedplaced

When an electric current is added to When an electric current is added to the gel at the location of the wells, the gel at the location of the wells, DNA starts to move to the opposite end DNA starts to move to the opposite end (+)(+)

Large DNA pieces take longer to move Large DNA pieces take longer to move through the gel than smaller piecesthrough the gel than smaller pieces

Standard Marker

Current applied here (-)

(+) PoleGel and buffer

PurposePurpose Separating DNA pieces based on Separating DNA pieces based on

sizesize Can be used to analyse PCR Can be used to analyse PCR

productsproducts Is used in DNA sequencingIs used in DNA sequencing To detect the presence/lack of a To detect the presence/lack of a

particular gene in a plasmidparticular gene in a plasmid

Restriction and Ligase Restriction and Ligase EnzymesEnzymes

Restriction:Restriction: Naturally occurring Naturally occurring Can cut DNA backbones at specific Can cut DNA backbones at specific

nucleotide sequences called nucleotide sequences called recognition recognition sequencessequences

Many different types, isolated from many Many different types, isolated from many different species of bacteriadifferent species of bacteria

Mostly cut DNA unevenly, creating Mostly cut DNA unevenly, creating ‘‘sticky endssticky ends’’

Ligase:Ligase: Also naturally occurringAlso naturally occurring Join DNA backbones togetherJoin DNA backbones together

Restriction Enzyme Restriction Enzyme PurposePurpose

Used to cut DNA Used to cut DNA at specific points at specific points for:for: Genetic Genetic

recombination recombination (inserting a gene (inserting a gene into a bacterial into a bacterial plasmid)plasmid)

Gel Gel electrophoresiselectrophoresis

EcoRI recognition sequence

Sticky End

Sticky End

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Uses principle of DNA replication Uses principle of DNA replication using DNA polymeraseusing DNA polymerase

Uses specific form of DNA Uses specific form of DNA polymerase called polymerase called TaqTaq polymerasepolymerase which is isolated which is isolated from bacteria that live in from bacteria that live in volcanic ventsvolcanic vents Therefore heat resistantTherefore heat resistant

What is needed?What is needed? Sample DNASample DNA Primers (complementary to Primers (complementary to

‘target sequence’ or DNA section ‘target sequence’ or DNA section of interest)of interest)

DNA nucleotidesDNA nucleotides TaqTaq polymerase polymerase

3 Steps…3 Steps… Denaturation Denaturation

heat up DNA sample to 94-96◦C heat up DNA sample to 94-96◦C DNA strands separate – these act as a DNA strands separate – these act as a

templatetemplate Annealing Annealing

Sample cooled to 50-65◦CSample cooled to 50-65◦C Primers bind to target sequencePrimers bind to target sequence

ExtensionExtension Temperature raised to 72◦CTemperature raised to 72◦C TaqTaq polymerase binds to primer/DNA polymerase binds to primer/DNA

strand and constructs a new strand that strand and constructs a new strand that is complementary to the original DNA is complementary to the original DNA

RepetitionRepetition The process is repeated or cycled The process is repeated or cycled

through many times:through many times: With every cycle, the amount of DNA With every cycle, the amount of DNA

is doubledis doubled Each cycle takes around 5 minutesEach cycle takes around 5 minutes Usually, the process is continued for Usually, the process is continued for

around 20-30 cyclesaround 20-30 cycles How many copies of DNA after 20 How many copies of DNA after 20

cycles? (2cycles? (22020)) How many after 30 cycles?How many after 30 cycles?

PurposePurpose•The purpose of PCR is to produce many copies of a particular sequence of DNA for:

•Creating recombinant plasmids•Detecting DNA of particular organisms

•So it is often called ‘amplification’

Thermal cycler used in automated PCR

Recombinant Plasmids & Recombinant Plasmids & transformation transformation

Refer to animationsRefer to animations

DNA Microarrays DNA Microarrays A type of technology used to A type of technology used to

detect mRNA produced by detect mRNA produced by certain cells certain cells

Therefore can analyse which Therefore can analyse which genes are activegenes are active

Can be used to diagnose some Can be used to diagnose some forms of cancer that are forms of cancer that are genetically inheritedgenetically inherited

Procedure Procedure To investigate protein production:To investigate protein production:

Isolate mRNA for genes and all of the Isolate mRNA for genes and all of the possible alleles that you would like to possible alleles that you would like to investigateinvestigate

Convert mRNA to DNA that is Convert mRNA to DNA that is complementary to the mRNA using an complementary to the mRNA using an enzyme called reverse transcriptase. The enzyme called reverse transcriptase. The process is called process is called reverse transcriptionreverse transcription and and the product is called copy DNA (the product is called copy DNA (cDNAcDNA) )

Attach the cDNA to a glass slide in Attach the cDNA to a glass slide in specific positions using a robotic machinespecific positions using a robotic machine

Record positions of each cDNA moleculeRecord positions of each cDNA molecule

Procedure Procedure Extract a sample of mRNA isolated from a Extract a sample of mRNA isolated from a

specific population of cellsspecific population of cells Convert to cDNA using reverse Convert to cDNA using reverse

transcriptase and PCRtranscriptase and PCR Label the sample cDNA with fluorescent Label the sample cDNA with fluorescent

dyesdyes Add cDNA sample to the glass slideAdd cDNA sample to the glass slide The cDNA in the sample will bind to The cDNA in the sample will bind to

complementary sequences on the slidecomplementary sequences on the slide The slide can then be viewed using a The slide can then be viewed using a

fluorescence microscopefluorescence microscope The colours seen can indicate that the The colours seen can indicate that the

cDNA has bound – mRNA must be in cDNA has bound – mRNA must be in sample so gene must be activesample so gene must be active

DNA MicroarrayDNA Microarray

DNA DNA profiling/fingerprintingprofiling/fingerprinting

http://www.biotechnologyonline.gov.http://www.biotechnologyonline.gov.au/popups/int_dnaprofiling.cfmau/popups/int_dnaprofiling.cfm

http://www.teachersdomain.org/resohttp://www.teachersdomain.org/resources/tdc02/sci/life/gen/creatednafinurces/tdc02/sci/life/gen/creatednafingerprint/index.htmlgerprint/index.html

http://www.pbs.org/wgbh/nova/shephttp://www.pbs.org/wgbh/nova/sheppard/lab74.htmlpard/lab74.html

http://http://www.bioloj.ca/biotech/dna_manipulawww.bioloj.ca/biotech/dna_manipulation/dna_profiling.htmltion/dna_profiling.html