Unit 7- Analysing DNA Flashcards
What is the purpose of PCR?
To make large number of copies of DNA fragments
In PCR why do we use primers? And why is the primer we chose important
- primers act as a binding point for DNA polymerase, as it can only bind to double stranded DNA
- only the DNA between primers is replicated, so we need to chose a primer than doesn’t overlap the gene that we are trying to replicate
Why isn’t DNA polymerase denatured at the high temps of PCR? And what’s this enzymes optimum temp?
- the DNA polymerase used derives from a thermophillic bacterium that lived in hot springs (90 degree)
- optimum temp is 72 degrees c
Describe the process of PCR
- solution of DNA fragments is heated to 90-95 degrees C for 30s
- cooled to 50-55 degrees C, primers bind
- heat to 72 degrees C for DNA polymerase to build complementary strand
In PCR why do we heat to 95 degrees, then add primers and cool to 50 degrees, and then reheat to 72 degrees?
95 degrees breaks hydrogen bonds
At 50 degrees hydrogen bonds can form between strand and primers
72 degrees is the optimum temp for DNA polymerase
In PCR after 3 repeats how many times more strands of DNA are there?
2^3
So 8, as its 2^n, when n is the number of times the process is repeated
Positive and negatives of PCR
+ can be used to make more from a tiny sample, useful in forensics
- the sample needs to be pure or contaminant DNA will also be amplified
What is the difference between PCR and Gene sequencing
Gene sequencing uses terminator bases with fluorescent tags and the sequence is worked out with computers
PCR replicates the sequence
Describe the process of gene sequencing
- samples of fragmented DNA are heated to 95 degrees C to break hydrogen bonds
- sample cooled to 50 so primers can anneal
- sample reheated to 72 degrees do DNA polymerase builds the strands until a terminator base stops the sequence
- sequence can be determined using by separating out fragments via gel electrophoresis or computers
What is a terminator base?
A nucleotide with an oxygen missing so the phosphodiester bond cannot form on one side so the sequence can’t continue
Why do we use DNA sequencing?
- to compare sequences between species to see how closely related
- to compare sequences between individuals to deduce family relation
- to identify allleles linked with genetic diseases
What is an issue with gene sequencing?
Predicting amino acid sequences is hard because introns are removed from DNA in splicing
What is DNA profiling used for?
- used in forensics and paternity tests
Explain what STRs and VNTRs are
Regions of non coding DNA that contain simple repetitive sequences
- short Tandem repeats (STRs) are when a sequence of about 4 nucleotides are repeated about 3-15 times
- everyone has STR sequences in the same loci but different people have different numbers or repeats
- STR regions are called (VNTR), variable number tandem repeats
Describe the process of DNA profiling
1- begin with the sample of DNA
2- amplify the sample of DNA via PCR using special primers that bind at the start of the VNTR sequence so the repetitive sequence is replicated
3- use restriction endonucleases to cut the DNA into unique fragments
4- seperate the fragments by electrophoresis
5- southern blot the agar
6- light emitted by fluorescent primers are detected by sensor
What can DNA profiling be used for?
- in forensics to match DNA samples to criminals
- paternity tests
- to prevent inbreeding in zoos
- to measure genetic diversity
- to establish phylogenetic relationships between species
What is electrophoresis?
Separating different pieces of DNA on the basis of their length using electric currents
Describe what happens in electrophoresis
- DNA samples are placed into wells at one end of the slab of the agar
- covered in a buffer solution
- an electric current is passed through the gel
- DNA is negatively charged due to the phosphate group so diffuses through the gel to the positive electrode
- the longer the strand of DNA the slower it diffuses
How can we make it so we can see the bands produced by electrophoresis??
- stain the DNA with azure which is blue
- stain the DNA with a fluorescent molecule (ethidium bromide) which emits colour under UV
What is the purpose of the southern blot?
To detect specific target sequences in a sample of DNA
What is a DNA probe?
A single Strand of DNA with radioactive/fluorescent label that will anneal to its complementary sequence
What is hybridisation?
When a DNA probe anneals to its complementary strand forming double stranded hybrid DNA
Describe the southern blot method
1- DNA is extracted and amplified by PCR
2- the dna is digested by restriction endonucleases to make smaller fragments
3- fragments are separated by gel electrophoresis after adding alkali to break the double stranded fragments into single stranded DNA
4- a sheet of nylon is placed on the gel, the DNA sticks to the nylon membrane
5- the nylon is placed in a solution of the labelled probes which will anneal to their complementary strands on the nylon forming hybrid DNA
6- the location of the hybrid DNA can be seen using a UV light as the labels may be fluorescent or auto radiography if radioactive labels were used
