BIOL5 Revision - joshdlx Watch
I'll be posting summaries of what I've revised here for AQA A-Level Biology Unit 5. The schedule will probably be:
Friday 17th June
- Gene Technology (starting with the hardest first )
Saturday 18th June
Sunday 19th June- Muscles and Muscle Contraction
- Muscles and Muscle Contraction
From Monday - Tuesday I'll be focusing on synoptic essays and past papers, so I might post some things here if I feel like it will help anyone. Wednesday I'll be quickly going through every topic again and doing more papers and essays.
Today I'll be revising gene technology. I split this topic up as follows:
1. Making DNA fragments
2. Gene cloning
3. Genetic engineering
4. Genetic fingerprinting
5. Locating and sequencing genes
6. Genetic counselling and gene therapy
Within each topic I'll post notes on sub-sections.
I am assuming basic knowledge from the previous topic on Genetics.
Why do we need DNA fragments?
• Gene technology is the study of genes
• It is necessary to have a DNA fragment containing the target gene that needs to be studied
Method 1 - Reverse Transcriptase
Most cells will only have two copies of each gene, which could make finding and extracting a DNA fragment containing the target gene difficult. However, remember that a cell will contain many mRNA molecules that are complementary to the target gene. Recall that from the Genetics topic, mRNA is used as a template to produce strands of cDNA (complementary DNA). The following are required to produce a DNA fragment using this method:
• Free DNA nucleotides
• mRNA complementary to the target gene
• The enzyme reverse transcriptase
The process is as follows:
1. Isolate and extract the mRNA that is complementary to the target gene from the cell
2. Mix the complementary mRNA, the free nucleotides and the enzyme reverse transcriptase
3. Reverse transcriptase will use the free nucleotides to produce a strand of cDNA (fragment contains the target gene)
Before we begin looking at this method, it is necessary to have knowledge of palindromic sequences.
What are palindromic sequences? They are sections of DNA with antiparallel base pairs that can be read the same in opposite directions.
Why are palindromic sequences important in this case? They're important because they can act as recognition sequences for enzymes known as restriction endonucleases. Restriction endonucleases will cut (digest) the DNA at a recognition sequences.
The specificity of restriction endonucleases must be noted. Each restriction endonuclease will only cut the DNA by hydrolysis at a specific palindromic sequence. This is because it is the shape of the palindromic sequence that is complementary to the shape of the active site of a specific restriction endonuclease enzyme.
The segment in between two palindromic sequences can be cut out - this is the DNA fragment.
The process is as follows:
1. Incubate the DNA sample with the specific restriction endonuclease(s)
2. The restriction endonuclease(s) will cut the DNA fragment out in a hydrolysis reaction
The last thing to note is that the DNA fragment produced might have some 'sticky ends'. However, this isn't a disadvantage. These sticky ends - which are unpaired bases - can be used to anneal another section of DNA to the DNA fragment, providing that this other section of DNA has sticky ends that are complementary to the sticky ends of the DNA fragment.
This is a lot easier than it might appear, and has a lot in common with the restriction endonuclease method. PCR is beneficial because it doubles the amount of DNA each time and can constantly be repeated to result in lots of DNA fragments within a short space of time.
What do you need for the PCR reaction?
• The sample of DNA containing the target gene
• Primers, which are short pieces of DNA complementary to the bases at the start of the fragment that you want (similar to restriction endonucleases) - note the specificity of primers
• DNA polymerase, which creates new strands of DNA
• Free nucleotides in order to allow for complementary base pairing and so the formation of new strands
The process is as follows:
1. Mix the DNA sample containing the target gene, the free nucleotides, primers and DNA polymerase.
2. Heat the DNA mixture to 95 degrees Celsius - this is done to break the hydrogen bonds between the two strands of DNA
3. Cool the mixture to around 65 degrees in order to allow the primers to anneal to the single strands of DNA
4. Heat the reaction mixture again to 72 degrees to allow DNA polymerase to function - the DNA polymerase will line up the free DNA nucleotides in the mixture alongside each template strand
5. Specific base pairing will occur, where new, complementary strands are formed (double-stranded)
Two new copies of the fragment of DNA have been produced.
The cyclical nature of PCR means that the process can repeat - a new mixture can be heated to 95 degrees. All four strands (the 2 original strands and the 2 new strands) can be used as templates. This is what means that so many DNA fragments can be produced. In summary: PCR is used to amplify DNA.
I signed up here to do one of these threads after I was inspired by Anon_98's. He will probably do one of his own for BIOL5, but I guess there can be two (as long as he doesn't mind!)