chapter 13: molecular techniques Flashcards
what is the polymerase chain reaction?
- it is a technique in which a specific sequence of DNA is amplified in a short period of time
- without use of a living organism
- PCR is in vitro ( in test tube) method and is a more rapid approach to cloning
- than cell based in-vivo ( in the living) method
MATERIALS REQUIRED FOR PCR
why is template DNA required for PCR? and where can the sample DNA be from?
- sample DNA contains target DNA sequence to be amplified and can be from any source
- the DNA sample is used as a template during PCR
- the only condition is that DNA sequences flanking the target DNA sequence to be amplified must be known to design primers
- or else is cannot be amplified using PCR
DNA sample may be:
- DNA form blood, semen or an tissue, like a strand of hair, a drop of blood
- DNA from older forensic specimens like fragment of ancient DNA from mammoths preserved in ice
- DNA from viruses in clinical samples and micro-organisms like bacterial colonies
MATERIALS REQUIRED FOR PCR
what are primers and why are they required for PCR?
- primers are short synthetic single-stranded DNA fragments which have complementary sequences to sequences on the sample DNA
- which flank the target DNA sequence to be amplified
- to amplify a particular sequence, two different primers are used
- both primers are designed with sequence in 5’ to 3’ direction so that each can anneal to the sequence flanking the target DNA sequence to be amplified at the 3’ ends of both template strands
- (it is annealed by forming CBP through hydrogen bonds
- primers provide free 3’-OH group of a pre-existing chain base paired to the template DNA for Taq DNA polymerase to add deoxyribonucleotides to
- ## primers also mark/ specify the region of DNA to be amplified
what is the difference between the primers in DNA replication and in PCR?
- the primers in DNA replication used are RNA
- whereas the primers in PCR are DNA
MATERIALS REQUIRED FOR PCR
what are the characteristics of Taq DNA polymerase? and what are some of its characteristics? ( compare to human DNA polymerase)
- it is a highly thermostable DNA polymerase isolated from a bacterium called Thermus aquaticus
- the bacteria live in hot springs at temperatures of up to 90°C
- Taq DNA polymerase has a similar function to human DNA polymerases
- Taq DNA polymerase is however more thermostable than human DNA polymerases and has an optimum temperature of about 70°C
- unlike human DNA polymerase, it does not lose its three-dimenasional configuration and is not denatured at high temperatures
-Taq DNA polymerase also lacks 3’ to 5’ exonuclease proofreading function unlike human DNA polymerases
- this results in low fidelity and causing relatively high error rates during replication of DNA in PCR
MATERIALS REQUIRED FOR PCR
what are the 4 deoxyribonucleoside triphosphates (dNTPs) and what are they used for
- dCTP, dATP, dGTP, dTTP
- are monomers used for the synthesis of new DNA strands
MATERIALS REQUIRED FOR PCR
what is the buffer solution for and what does it contain?
- it prevents pH changes which will interfere with enzyme action
it also contains:
1. MgCl2, Mg2+ ions which functions as a cofactor for DNA polymerases like Taq polymerase
- it also helps facilitate the bidning of primers to the DNA template
- it will form complexes with the deoxyribonucleotides, primers and DNA
- and optimise conditions for taq DNA polymerase, ensuring efficient and specific DNA amplification
- high grade water
- the water is free from RNA, DNA or nucleases which may contaminate or degrade primers or template DNA strands - mineral oil
- prevents drying out of mixtures over the period of PCR cycles because it will be subjected to high temp
-
READ:
- all the materials are mixed in a small test tube and placed in a thermal cycler
- PCR is carried out using an automated thermal cycler
- the thermal cycler is a sophisticated heating block that is able to change temperature rapidly over short time intervals
- the thermal cycler takes the sample through a series of reactions called a PCR cycle
nothing!
what are the steps of the PCR cycle?
step 1: denaturation of DNA
- reaction mixture is heated to about 94°C to 96°C
- hydrogen bonds between complementary bases of the double-stranded template DNA are broken
- the double-stranded DNA is denatured, causing the two strands to separate forming two single-stranded DNA
step 2: annealing of primers
- reaction mixture is cooled to 54°C to 60°C
- primers bind to complementary sequences at the 3’ ends of both strands of the template DNA which flank the target DNA sequence to be amplified
step 3: extension of primers
- reaction mixture is heated to 72°C
- (which is the optimum temperature for taq DNA polymerase
- which will allow the maximum rate of addition of DNA nucleotides and the formation of phosphodiester bonds)
- taq DNA polymerase extends the 3’ ends of both primers by adding deoxyribonucleotides to the free 3’ OH ends of primers, synthesising double stranded DNA molecules
- the order of bases is determined by CBP with the template DNA strand
- each three step cycle is then repeated _ to _ times
- each time the target DNA sequence is copied, it serves as a ____ DNA for the next cycle
- each repeat of the cycle results in amount of DNA being ____
- one cycle of denaturation, annealing and extension can take several minutes and thus billions of copies of a specific target DNA sequence can be produced in a few hours
- each three step cycle is then repeated 30 to40 times
- each time the target DNA sequence is copied, it serves as a template DNA for the next cycle
- each repeat of the cycle results in amount of DNA being doubled
- one cycle of denaturation, annealing and extension can take several minutes and thus billions of copies of a specific target DNA sequence can be produced in a few hours
what are the three advantages of PCR?
- it is fast and easy to use
- high specificity
- high sensitivity
advantages of PCR
why is being fast and easy an advantage?
- a typical PCR run can amplify large amount/ many copies of DNA in a short time and the proccess is automated
read:
this is more efficient than cell-based DNA cloning which may take several days and also require clean-up of unwanted cellular debris
advantages of PCR
why is high specificity an advantage of PCR? and what is it due to?
- PCR is a highly specific process that amplifies a specific region of DNA
- this specificity is due to the primers which bind to specific sequences at 3’ ends flanking the target sequence via complementary base pairing
- for high specificity, the primers must be at least 15 nucleotides long
advantages of PCR
why is high sensitivity of PCR an advantage?
- using only a small amount of the sample DNA containing the complete target sequence, a specific DNA sequence can be amplified to large amoun
- due to high sensitivity of PCR, it can be used to amplify specific DNA sequences where sample amounts are limited
- eg. DNA from crime scene in forensic science or badly degraded DNA like DNA recovered from archaelogical remains
what are the 4 limitations of PCR?
- low replication fidelity
- prone to contamination
- target DNA sequence information needed
- length limitation
