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Description
Flashcards by sophiakostich, updated more than 1 year ago
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Created by sophiakostich
almost 11 years ago
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| Question | Answer |
| What are two of the main techniques that can be used in molecular diagnostics? | PCR and southern/northern blotting |
| What are the three simple steps which describe how diseases can be diagnosed by analysis of DNA from a patient? | Extract DNA from patient, Break DNA into fragments (not always necessary), test for specific (disease associated) DNA sequences |
| What is the function of restriction enzymes? | To break DNA into fragments normally of 4-6 bp by recognising and cleaving at specific DNA sequences. |
| What are the two types of ends that can result from cutting with restriction enzymes? | Blunt or sticky |
| What does it mean when it says restriction enzyme cutting sites are pallindromic? | The top and bottom strands have the same sequence forwards and backwards e.g. top strand AATT then bottom strand is TTAA |
| Give 2 examples of restriction enzymes. | Eco RI and Hae III |
| Does Eco RI cleave DNA to sticky ends or blunt ends? | Sticky. |
| What is the size of the target site recognised by Eco RI? | 6bp |
| What is the sequence that Eco RI recognises? | GAATTC |
| How large would the fragments produced by Eco RI be if it cut human DNA? How many fragments would be produced if it digested human DNA? | Fragments would be 4096 bp therefore 750,000 would be produced as 3 x 10^9 (size of human genome) divided by 4096 = 750,000 |
| What is the function of gel electrophoresis? | To separate DNA fragments of different sizes |
| Which end do DNA and RNA migrate to in gel electrophoresis? | The positive end of the gel. |
| Which migrate faster in gel electrophoresis: large or small fragments? | Small fragments as the gel acts like a sieve. |
| Does the restriction enzyme Hae III produce blunt ends or sticky ends? | Blunt |
| How can DNA/RNA fragments be seen as bands on the gel? | By treating the gel with a stain. |
| What is used in southern blotting to show the fragment of DNA that is needed following electrophoresis? | Southern blotting uses a radioactively labelled probe that can base pair with specific DNA fragments in the mixture. |
| What are the main steps in southern blotting? | Restriction enzymes, electrophoresis, treat agarose gel with NaOH to denature DNA, blot DNA onto nylon membrane, boil radioactive probe, incubate probe with membrane, probe base pairs with DNA, expose to X-ray film. |
| How does the DNA from the gel blot onto the nylon membrane? | The membrane is placed between filter paper soaked in buffer, gel and tissue paper and left overnight. The buffer moves through the gel and towards the stacks of tissue paper creating a flow of DNA. |
| What is it important to do after the probe and nylon membrane are incubated overnight? | To wash the membrane to remove any unbound probe. |
| What is the restriction enzyme used in diagnosis of sickle cell via southern blotting? | Mst II |
| What is it callled when digestion of normal beta-globin genes and mutated ones (in sickle cell) with Mst II produces fragments of different sizes? | Restriction fragment length polymorphism |
| Is northern blotting the same or different to southern blotting? | Overall it is fairly similar apart from electrophoresis step is used to fractionate RNA and not DNA. |
| What is the function of PCR? | Targeted amplification of a specific DNA fragment. |
| What are three circumstances where PCR may be used? | Diagnosis and detection of carriers, diagnosis of cancer and diagnosis of infectious diseases/pathogens |
| What are two of the key components of PCR? | Primers and heat stable DNA polymerase enzyme - Taw DNA polymerase |
| True or false: two primers are needed in PCR - a forward and a reverse. | TRUE |
| What are the four things that a PCR reaction contains? | Small amounts of DNA to be amplified, two primers, dNTPs, Taq DNA polymerase |
| In most cases how many cycles of amplification occur? How long may this take? | 20-40, 2-3 hours |
| What are the three main steps in PCR? | 1. Incubation at 95 degrees to denature DNA 2. Incubation at 45-60 degrees to allow primers to stick to single stranded DNA 3. Incubation 72 degrees - optimum temp for Taq DNA polymerase |
| What are the three main steps in PCR? | 1. Incubation at 95 degrees to denature DNA 2. Incubation at 45-60 degrees to allow primers to stick to single stranded DNA 3. Incubation 72 degrees - optimum temp for Taq DNA polymerase |
| True or false: the amount of target fragment triples during each cycle there after n cycles there will be 3^n copies of the gene. | FALSE. After each cycle the amount of fragment doubles so there will be 2^n copies. |
| What kind of mutation occurs in huntingtons disease? Give examples. | Trinucelotide expansion. Normal - CAG (15-29). Typical huntingtons - CAG (40-60) Early onset - CAG (more than 60) |
| When can PCR be used to show that someone has the huntingtons gene? | In pre-natal diagnosis and in adults with affected parents |
| What are the three main steps involved when PCR is used? | Isolate DNA --> PCR --> electrophoresis |
| What form of PCR is often used to diagnose/find bacteria and viruses? Why and how? | Real time PCR. Because it skips to the electrophoresis step and can be done very quickly by adding dye that fluoresces when it binds to double stranded DNA (fluorescence correlates to no. of cycles) |
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