Spec statements: Fragments of DNA can be produced by

1. Spec statements: Fragments of DNA can be produced by • Conversion of mRNA to cDNA, using reverse transcriptase. • Cutting DNA at specific, palindromic recognition sequences using restriction endonucleases. • The polymerase chain reaction (PCR) Fragments of DNA produced by the above methods can be used to clone genes by in vivo and in vitro techniques.

2. Gene Cloning Lesson 1

3. Outcomes -Describe a brief outline of the steps involved in gene cloning -List the 3 ways of producing fragments for gene cloning -Compare the techniques of using reverse transcriptase and restriction endonucleases.

4. An overview of Gene Cloning • https://www.youtube.com/watch?v=dRW9jIOdBcU • Watch out for… • the use mentioned in the video • the organism involved • the steps involved • Now summarise the process of gene cloning in no more than xxx words

5. Todays lesson

6. Isolating the target gene • Aim-you would like a fragment of DNA that you can insert into the organism. Fragments of DNA can be produced by: -reverse transcriptase -restriction endonucleases -PCR

7. Reverse Transcriptase • Does what it says on the tin! • Coverts mRNA into cDNA (single stranded) • Naturally found in retro viruses such as HIV. Retrovirus have RNA as their genetic material. They contain reverse transcriptases so that DNA can be produced that can then be transcribed by the cell that they have infected. • Advantages-most cells do not transcribe all of their genes = less mRNA in a cell than DNA. It is therefore easier to find and isolate the gene you want from mRNA.

8. Restriction Endonucleases. • Nucleases are enzymes that break the bond linking one nucleotide to another in a DNA strand. • Restriction endonucleases are enzymes that break the bonds in the sugar phosphate backbone of both strands of the DNA. • They make the cut at palindromic recognition sequences.

9. Palindromic Sequences. • A sequence of DNA that reads the same from both the 5’ end and the 3’ end. • Why is it that the endonuclease will only cut at the palindromic sequence? • They are enzymes-their active site has a specific shape –BIOL 1.

10. Restriction Endonucleases. More Questions • When using an RE, how can scientist be sure that they will be only cutting out one gene? • They need to know the base sequence either side of the gene, make sure that this is palindromic and that this is unique to this gene. • RE’s are found naturally in bacteria such as ECO R1 which is found in E.Coli. Bacteria use it to fight off bacteriophages. Why do the RE’s not act on the bacteria? • Because they do not contain the palindromic sequence that it acts on.

11. Restriction Endonucleases. Last point….there are 2 types. 1. Produces blunt ends. Cuts the DNA at exactly the same base number. Produces sticky ends. Does not cut at the same base number. This is the type used for gene cloning.

12. Tasks…. • Complete key terms sheet. • Make a list of at least 3 comparisons between reverse transcriptases and restriction endonucleases. • Answer the question… Describe how DNA fragments can be isolated for cloning. 6 marks.

14. Made up mark scheme Describe how DNA fragments can be isolated for cloning. 6 marks Any 6 of the following Enzymes Reverse transcriptase Turns mRNA into cDNA. Gene can be isolated from mRNA Less mRNA than DNA Restriction endonuclease Cuts at specific recognition sequence Specific due to shape of active site ECO R1 example Produces sticky ends.

15. Isolating the target gene • Aim-you would like a fragment of DNA that you can insert into the organism. Fragments of DNA can be produced by: -reverse transcriptase -restriction endonucleases -PCR Next lesson. Read up on this for homework.