Genetic Fingerprinting Flashcards
What is a genetic fingerprint?
Different from the DNA sequence bc it only shows non-coding portions of DNA. uses the PCR to make large numbers of it and gel electrophoresis to separate DNA fragments by size.
What is the DNA of a genetic profile?
Exons code for proteins and introns are within the base sequences not coding for proteins. Introns have sequences where up to 13 bases repeat up to several hundreds of times.
What are short tandem repeats?
The place on introns where up to 13 bases repeat up to 100s of times. The number of repeats within an STR is different for everyone so is unique and therefore makes up the genetic fingerprint.
What does PCR do?
Semi-conservative replication of DNA, working rapidly and amplifies the amount of DNA.
What is the DNA sample dissolved in and mixed with In PCR?
A buffer and mixed with taq polymerase, nucleotides containing the 4 DNA bases, and short single stranded bases of DNA called primers between 6-25 bases long.
What is taq polymerase?
DNA polymerase from a bacterium that lived in hot springs with high optimum temps.
What are primers?
Short single stranded bases of DNA complimentary to the start of the DNA strand and binding to it, signalling taq polymerase to start replication
What are the 4 stages of PCR?
- Target DNA heated to 95 degrees so it separates to 2 strands
- Solution cooled to 55 degrees so primers anneal to single strands of DNA
- Heated to 70 degrees and taq polymerase catalyses synthesis of a complimentary strand by adding complimentary nucleotides (elongation extension phase).
- Two identical strands produced. Repeated many times.
What are 5 limitations of PCR?
- Contamination
- Error rate
- DNA Fragment size
- Sensitivity to inhibitors
- Limits on amplification.
How can PCR contaminate DNA?
Any contaminating DNA from the investigator or any air bourne DNA will be amplified.
How is the error rate a limitation of PCR?
DNA polymerase sometimes inserts the wrong base. Taq polymerase can’t proofread the DNA so it makes an error. The cycles repeat this error.
How is DNA fragment size a limitation of PCR?
PCR is efficient at making DNA 1000-3000 bases long because taq polymerase can’t correct it’s errors. If a lower temperature higher pH and proofreading polymers are used a length of 40,000 base pairs can be generated. Many genes are much longer than this.
How is sensitivity to inhibitors a limitation of PCR?
molecules may act as inhibitors that PCR is sensitive to such as phenolics and haem breakdown products.
How is limits of amplification a limitation of PCR?
at the start the DNA strand increases exponentially but after 20 cycles it slows down and eventually plateaus because the Reagant conc can be limiting, the enzyme denatured and the DNA in high concentrations cause the single stranded DNA to base pair with each other not primers.
Give the stages of gel electrophoresis.
- DNA extracted and cut into thousands of fragments using restriction endonucleases
- Fragments seperated on an agarose gel (has pores)
- Samples loaded into wells and a voltage is applied. Phosphate groups of backbone have a neg charge so are attracted to the anode.
- Smaller fragments move more easily so migrate further.
- If fragments of known length are seperated on the same gel at the same time making a ‘DNA ladder’, the lengths of fragments can be estimated
- Covered with a nylon membrane which picked up the fragments (southern blotting)
- Radioactive or luminescent DNA probes attach by base pairing to the STRS and unbound ones washed off
- Film sensitive to X-rats or the wavelengths emitted by luminescent probes placed over blot overnight
- Film exposed and the autoradiograph shows the banding pattern showing sequences.
