Enzyme names to learn 1.Reverse transcriptase 2.RNA polymerase 3.DNA helicase 4.DNA ligase 5.DNA polymerase 6.Restriction endonuclease A.Unwinds DNA helix within the molecule B.Joins nucleotides in DNA replication C.Joins RNA nucleotides to make mRNA during transcription D.Obtained from retroviruses. Used to make DNA from mRNA E.Obtained from bacteria. Cut genes from DNA at specific recognition sequences F.Joins/splices genes to make recombinant DNA

Enzyme names to learn – correct order 1.Reverse transcriptase 2.RNA polymerase 3.DNA helicase 4.DNA ligase 5.DNA polymerase 6.Restriction endonuclease A.Obtained from retroviruses. Used to make DNA from mRNA B.Joins RNA nucleotides to make mRNA during transcription C.Unwinds DNA helix within the molecule (separates two strands) D.Joins/splices genes to make recombinant DNA E.Joins nucleotides in DNA replication F.Obtained from bacteria. Cut genes from DNA at specific recognition sequences

Syllabus Genes can be cloned by in vivo and in vitro techniques. In vivo cloning. The use of restriction endonucleases and ligases to insert DNA fragments into vectors, which are then transferred into host cells. The identification and growth of transformed host cells to clone the desired DNA fragments. The importance of “sticky ends”. In vitro cloning. The use of the polymerase chain reaction (PCR) in cloning DNA fragments. The relative advantages of in vivo and in vitro cloning

How do we make enough DNA fragments? Cloning means replicating them identically What is the difference between cloning in vivo and in vitro?

In vitro gene cloning – the polymerase chain reaction Aqa p.254-5

Objectives What is the polymerase chain reaction? How does the process work? Advantages of in vitro and in vivo gene cloning

Producing multiple copies of DNA

PCR headings Introduction (what PCR stands for and what it is for) Ingredients needed Process step 1 (p.254) Process step 2 Process step 3 ics/PCR/PCR.htm Advantages of in vitro and in vivo cloning

Exemplar work See ppt Next slide

Worksheet PCR

Advantages of in vitro and in vivo gene cloning in vitro = PCRin vivo = using bacteria

In the past, one of the drawbacks in obtaining genetic fingerprints from material present at a crime scene was the very small quantities of DNA recoverable for analysis A technique called the polymerase chain reaction was developed in 1983 by Kary B. Mullis providing the breakthrough that allowed scientists to produce multiples copies of a DNA sample within a very short period of time The polymerase chain reaction (PCR) mimics nature’s way of replicating DNA and is able to generate billions of copies of a DNA sample within a few hours - the technology allows for cheap and rapid amplification of DNA The technique involves heating DNA to high temperatures to separate the strands and then using the enzyme DNA polymerase to create new strands Due to the high temperatures required for the technique, a thermostable DNA polymerase had to be found to avoid the expense of needing to replenish the enzyme after each round of DNA replication The Polymerase Chain Reaction (PCR)

Animation – click below to open The Polymerase Chain Reaction (PCR) Animation – click below to open Click me, go on.

The solution to this problem was to use Taq polymerase, derived from Thermus aquaticus, a bacterium that is native to hot springs – this enzyme is able to withstand the high temperatures (up to 95 ° C) used in the polymerase chain reaction

The target DNA is first mixed with DNA polymerase and primers and then heated to 95 ° C to separate the two strands of DNA The Technique Primers are short, synthetic DNA fragments that are complementary to the DNA sequences at either end of the region of DNA to be copied

The Technique The mixture is now cooled to 55 ° C to allow the primers to bind to the ends of the separated DNA strands Polymerase binds to the primers and begins adding bases to form new complementary strands

The Technique The mixture is now cooled to 55 ° C to allow the primers to bind to the ends of the separated DNA strands Polymerase binds to the primers and begins adding bases to form new complementary strands

Two Identical Copies of the Target DNA Sequence Result From the First Synthesis Cycle

The process is now repeated by first heating the mixture to separate the strands of the newly formed DNA molecules The sample is cooled to allow the primers to attach to the ends of the DNA strands so that polymerase can begin its job of adding bases to the sequence

At the end of the second cycle there are four complete DNA molecules identical to the original target DNA Cycle 2 Products The cycle is repeated many times with the number of DNA molecules doubling with each cycle This exponential increase creates over a billion copies of the target DNA within a few hours

Cycle 2 Products Cycle 3 Products The number of DNA molecules doubles with each cycle

Target DNA is heated to separate the strands When the mixture is cooled, primers bind to the ends of the target strands and polymerase enzymes add bases to complete the complementary strands Two identical DNA molecules are formed A second cycle is initiated by heating the mixture once again to separate the strands of the newly formed DNA molecules When the mixture is cooled, primers bind to the ends of the target strands and polymerase enzymes add bases to complete the complementary strands Four identical copies of the target DNA are formed at the end of the second cycle This cycle of heating and cooling continues for approximately 30 cycles, doubling the number of DNA molecules with each cycle SUMMARY PCR generates billions of copies of target DNA within a few hours