Molecular Genetic Technologies Gel Electrophoresis PCR Restriction & ligation Enzymes Recombinant plasmids and transformation DNA microarrays DNA profiling

Gel Electrophoresis DNA is negatively charged DNA is negatively charged Uses an agarose gel which contains tiny wells in which DNA samples can be placed Uses an agarose gel which contains tiny wells in which DNA samples can be placed When an electric current is added to the gel at the location of the wells, DNA starts to move to the opposite end (+) When an electric current is added to the gel at the location of the wells, DNA starts to move to the opposite end (+) Large DNA pieces take longer to move through the gel than smaller pieces Large DNA pieces take longer to move through the gel than smaller pieces

Standard Marker Current applied here (-) (+) Pole Gel and buffer

Purpose Separating DNA pieces based on size Separating DNA pieces based on size Can be used to analyse PCR products Can be used to analyse PCR products Is used in DNA sequencing Is used in DNA sequencing To detect the presence/lack of a particular gene in a plasmid To detect the presence/lack of a particular gene in a plasmid

Restriction and Ligase Enzymes Restriction: Restriction: Naturally occurring Naturally occurring Can cut DNA backbones at specific nucleotide sequences called recognition sequences Can cut DNA backbones at specific nucleotide sequences called recognition sequences Many different types, isolated from many different species of bacteria Many different types, isolated from many different species of bacteria Mostly cut DNA unevenly, creating ‘sticky ends’ Mostly cut DNA unevenly, creating ‘sticky ends’ Ligase: Ligase: Also naturally occurring Also naturally occurring Join DNA backbones together Join DNA backbones together

Restriction Enzyme Purpose Used to cut DNA at specific points for: Used to cut DNA at specific points for: Genetic recombination (inserting a gene into a bacterial plasmid) Genetic recombination (inserting a gene into a bacterial plasmid) Gel electrophoresis Gel electrophoresis EcoRI recognition sequence Sticky End

Polymerase Chain Reaction (PCR) Uses principle of DNA replication using DNA polymerase Uses principle of DNA replication using DNA polymerase Uses specific form of DNA polymerase called Taq polymerase which is isolated from bacteria that live in volcanic vents Uses specific form of DNA polymerase called Taq polymerase which is isolated from bacteria that live in volcanic vents Therefore heat resistant Therefore heat resistant

What is needed? Sample DNA Sample DNA Primers (complementary to ‘target sequence’ or DNA section of interest) Primers (complementary to ‘target sequence’ or DNA section of interest) DNA nucleotides DNA nucleotides Taq polymerase Taq polymerase

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 template DNA strands separate – these act as a template Annealing Annealing Sample cooled to 50-65◦C Sample cooled to 50-65◦C Primers bind to target sequence Primers bind to target sequence Extension Extension Temperature raised to 72◦C Temperature raised to 72◦C Taq polymerase binds to primer/DNA strand and constructs a new strand that is complementary to the original DNA Taq polymerase binds to primer/DNA strand and constructs a new strand that is complementary to the original DNA

Repetition The process is repeated or cycled through many times: The process is repeated or cycled through many times: With every cycle, the amount of DNA is doubled With every cycle, the amount of DNA is doubled Each cycle takes around 5 minutes Each cycle takes around 5 minutes Usually, the process is continued for around cycles Usually, the process is continued for around cycles How many copies of DNA after 20 cycles? (2 20 ) How many copies of DNA after 20 cycles? (2 20 ) How many after 30 cycles? How many after 30 cycles?

Purpose 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 & transformation Refer to animations Refer to animations

DNA Microarrays A type of technology used to detect mRNA produced by certain cells A type of technology used to detect mRNA produced by certain cells Therefore can analyse which genes are active Therefore can analyse which genes are active Can be used to diagnose some forms of cancer that are genetically inherited Can be used to diagnose some forms of cancer that are genetically inherited

Procedure To investigate protein production: To investigate protein production: Isolate mRNA for genes and all of the possible alleles that you would like to investigate Isolate mRNA for genes and all of the possible alleles that you would like to investigate Convert mRNA to DNA that is complementary to the mRNA using an enzyme called reverse transcriptase. The process is called reverse transcription and the product is called copy DNA (cDNA) Convert mRNA to DNA that is complementary to the mRNA using an enzyme called reverse transcriptase. The process is called reverse transcription and the product is called copy DNA (cDNA) Attach the cDNA to a glass slide in specific positions using a robotic machine Attach the cDNA to a glass slide in specific positions using a robotic machine Record positions of each cDNA molecule Record positions of each cDNA molecule

Procedure Extract a sample of mRNA isolated from a specific population of cells Extract a sample of mRNA isolated from a specific population of cells Convert to cDNA using reverse transcriptase and PCR Convert to cDNA using reverse transcriptase and PCR Label the sample cDNA with fluorescent dyes Label the sample cDNA with fluorescent dyes Add cDNA sample to the glass slide Add cDNA sample to the glass slide The cDNA in the sample will bind to complementary sequences on the slide The cDNA in the sample will bind to complementary sequences on the slide The slide can then be viewed using a fluorescence microscope The slide can then be viewed using a fluorescence microscope The colours seen can indicate that the cDNA has bound – mRNA must be in sample so gene must be active The colours seen can indicate that the cDNA has bound – mRNA must be in sample so gene must be active

DNA Microarray

DNA profiling/fingerprinting s/int_dnaprofiling.cfm s/int_dnaprofiling.cfm s/int_dnaprofiling.cfm s/int_dnaprofiling.cfm 02/sci/life/gen/creatednafingerprint/index.htm l 02/sci/life/gen/creatednafingerprint/index.htm l 02/sci/life/gen/creatednafingerprint/index.htm l 02/sci/life/gen/creatednafingerprint/index.htm l 74.html 74.html 74.html 74.html n/dna_profiling.html n/dna_profiling.html n/dna_profiling.html n/dna_profiling.html