Molecular Techniques & Diagnosis of DNA Flashcards
Describe the action of specific endonucleases
- Recognise and cut DNA at specific restriction sites
- Cut mostly palindrome DNA sequences
- Can produce ‘sticky ends’
- Separation of DNA into fragments
How does a bacteria protect its own DNA from degradation?
- Bacteria produce endonucleases
- METHYLATION of DNA protects DNA from restriction enzymes
What are ‘sticky ends’?
- Produced by restriction enzymes
- Staggered cut DNA with exposed unpaired DNA nucleotides at the ends
- Unpaired free nucleotides can form H bonds and come together
How can restriction enzymes help in the analysis of DNA at the gene level?
Can TARGET and ISOLATE specific genes to undergo further analysis which may be of clinical significance
Describe the method of DNA electrophoresis
- SEPARATES DNA ON BASIS OF SIZE
- DNA is cut into fragments by restriction enzymes and place on a gel
- Electric field applied forming +ve and -ve ends
- DNA migrates towards +ve end (anode) as it is -ve
- Speed and distance of travel depends on size of fragment
What are the main requirements for gel electrophoresis?
- Gel - matrix that allows separation of fragments
- Buffer - ionised liquid that allows charge of molecules across gel
- Power supply - generates charge difference across gel
- Stain - detect fragments
Why do we use restriction analysis?
- Investigate size of DNA fragments
- Investigate mutations (locate point mutations that may cause disease)
- Clone DNA
- Investigate DNA variation (fingerprinting)
Explain how a gene can be cloned with the assistance of a vector (bacterial plasmid)
- Gene targeted and isolated from DNA using restriction enzyme
- Plasmid cut open at specific restriction sites
- Gene is inserted into plasmid forming complementary H bonds with sticky ends and phosphodiester bonds using DNA LIGASE enzyme
- Plasmid inserted into bacteria which replicate producing many copies of gene
What is recombinant DNA?
DNA which contains genes/fragments from 2 different organisms e.g. Bacterial plasmid containing human gene for insulin
Explain the significance of bacterial conjugation
- Can share sections of DNA between two bacteria
- Antibiotic resistance genes can be shared
Describe the synthesis of proinsulin by gene cloning using bacteria
- Isolate human insulin gene in the form of mRNA
- Convert mRNA back to DNA using REVERSE TRANSCRIPTASE forming cDNA
- Join cDNA into bacterial plasmid and insert plasmid into bacteria
- Bacteria replicate and transcribe/translate gene producing insulin
What are the advantages of gene cloning?
- Make useful proteins e.g. Insulin
- Identify the action of genes e.g. how they are expressed and controlled
- Genetic screening for specific diseases e.g. Huntingdon’s, CF
- Gene therapy
What is the purpose of the polymerase chain reaction PCR?
- AMPLIFICATION of a gene/section of DNA
- Investigate single base mutations/insertions or deletions
- Investigate genetic relationships and variation
Explain the process of PCR in the amplification of a gene
- dsDNA fragments heated to 95 degrees breaking H bonds between bases (ssDNA formed)
- Cooled to 55 degrees and PRIMERS are added, which anneal to the 5’ end forming short double stranded sections of DNA on the ssDNA ends
- Heated to 72 degrees and taq polymerase is added - synthesises dsDNA using free dNTPs which bind complementary to ssDNA
- Process is repeated many times and copies of gene increase exponentially
State 3 ways in which we can analyse DNA at the gene level
- Restriction enzymes
- DNA electrophoresis
- PCR
What method can be used to analyse DNA at the sequence level?
DNA sequencing using Sanger dideoxy chain termination method
Describe the method of SOUTHERN BLOTTING
- DNA electrophoresis
- Fragments on gel transferred to a nitrocellulose/nylon sheet
- Sheet dipped in alkaline solution to break H bonds (forms ssDNA)
- DNA probes added with allele specific oligonucleotides which are complementary to gene on ssDNA
- Can locate specific sequences on DNA fragments
What is meant by DNA hybridisation and how is it used in Southern blotting?
- Two ssDNA molecules reanneal and form H bonds between complementary bases to form a dsDNA fragment
- In southern blotting a DNA probe with a fluorescent marker can reanneal complementary to one of the single strands so can be detected
What is the difference between Northern blotting and Southern blotting?
Northern blotting have follows the same process as Southern blotting but RNA is used instead of DNA
Describe the process of microarray
- Screens submicroscopic chromosome duplications or deletions
- Array of probes covering the entire genome
- Different coloured probes used for patient DNA and control DNA (normal)
- Patient DNA is RED, control is GREEN
- GREEN > RED means deletion
- GREEN
