7. ENZYME & RESTRICTION MAPPING Flashcards
What are some of the application of Genetic engineering?
Restriction enzymes can be used to:
- Produce recombinant proteins such as insulin, interferon, G-CSF
- Produce transgenic organisms & plants
What do nucleases do?
Nucleases degrade nucleic acids by hydrolyzing the phosphodiester bonds between the nucleotides
What are the two types of nucleases?
- Ribonucleases - degrade RNA
- Deoxyribonucleases - degrade DNA
* There are two types deoxyribonucleases:
- - >EXONUCLEASES - cleave the end of the sequence
- -> ENDONUCLEASES - cleave within the sequence
*Where do restriction enzymes/endonucleases originate from?
- Restriction enzymes are originally found/produced in bacteria. Different types of bacteria will produce different restriction enzymes
- Infecting phages or bacteriophages inject their nucleic acids into bacteria, so bacteria have restriction enzymes to counteract this.
The bacteria cleave the nucleic acids, limiting their transfer - Restriction enzymes in bacteria only cleave foreign DNA not their own because bacteria’s DNA is methylated
What do restriction enzymes do?
- Recognise specific sequences
2. Cut/cleave the sequence by hydrolysing phosphodiester bonds between the nucleotides
**What are recognition sites?
- Recognition sites are 4-8 bases in length, depending on the restriction enzyme
- They are palindromic meaning they can be read in either direction (5’3’ or 3’5’)
- Recognition sequence of 4 bases = 4^4 = 256
What are examples of restriction enzymes?
- EcoRI
- BamHI
- HindII
What are two outcomes of restriction enzyme cleavage?
- Blunt ends
2. Overhang - produced when phosphodiester bonds are cleaved
Give examples of nucleases which produce an overhang & blunt ends?
- EcoRI produces a 5’ overhang
- KpnI leaves a 3’ overhang
- AluI produces blunt ends
What are three uses of restriction enzymes?
- Cloning
- Molecular Diagnosis
- Inserting genes into plasmid/characterisation of plasmids
How can single nucleotide changes like SCD affect recognition sites?
- A single nucleotide change can change the recognition site
- E.g The restriction enzyme DdeI recognises the sequence CTGAG.
- BUT… in SCD, a single base substitution changes GAGA to GTG so the sequence now reads CTGTG.
- The recognition site is lost & DdeI cannot recognise the sequence
- This will show up as a missing site on PCR
What are restriction maps?
- Restriction maps are a visual representation showing the restriction sites for enzymes
What five enzymes are involved in forming recombinant DNA?
- DNA LIGASE
- DNA POLYMERASE
- PHOSPHATASE
- POLYNUCLEOTIDE KINASE
- REVERSE TRANSCRIPTASE
What does DNA ligase do in genetic engineering?
- DNA LIGASE is a ligating enzyme which joins the two fragments of DNA together.
- It makes new phosphodiester bonds between the cleaved sites
Why is DNA Polymerase used in genetic engineering?
- DNA Polymerase synthesises DNA from 5’ to 3’.
- DNA polymerase is needed for the blunt ending of the overhangs
- The overhang acts as a primer, so when DNA polymerase is added with nucleotides it can extend the overhang to produce a blunt end (double stranded)
What do phosphatases do in genetic engineering & why are they used?
- Phosphatases remove phosphates by hydrolyzing the phosphate group of a substrate
- Calf intestinal alkaline substrate or shrimp alkaline substrate can be used
- Phosphatases are needed to prevent cut plasmids from reannealing
What does a polynucleotide kinase do in genetic engineering & why are they used?
- Polynucleotide kinases add a phosphate to the 5’ hydroxyl of the DNA or RNA using ATP
- Chemically synthesised DNA isn’t phosphorylated, so it needs to be phosphorylated before it can be ligated & cloned
- Polynucleotide kinase also helps label DNA by using radioactive or fluorescent ATP
What does reverse transcriptase do in genetic engineering?
- Reverse transcriptase is a RNA dependent DNA polymerase which is isolated from retroviruses containing RNA
- It synthesises cDNA from the mRNA template using dNTPs
What three primers can be used in reverse transcription?
- Random
- Oligo (dT)
- Gene specific
*How is a random primer used in reverse transcription?
- A random primer is a 6 nucleotides long with any composition, but it binds to the sequence with perfect complementarity
- cDNA will be made in fragments
- But some very long mRNAs won’t be transcribed
How is an Oligo(dT) primer used in reverse transcription?
- An Oligo (dT) primer is a string of deoxythymidic bases (poly T tail). This poly T tail will act as a primer from DNA synthesis by binding to the poly A tail of mRNA
- However, if the poly T tail is too long, the reverse transcriptase won’t reach
the end of the mRNA template, and the whole DNA won’t be synthesized
How is a gene specific primer used?
- A gene spefic primer is found on an open, accessible region of DNA.
- It enhances sensitivity by directing the reverse transcriptase to a specific region rather than transcribing everything
- The reverse transcriptase is therefore more likely to reach the end of the mRNA template.
