Polymerase Chain Reaction (PCR) - BCH361 Practical PDF

Polymerase chain reaction (PCR) BCH361- Practical 2 § In a crime scene, a sample of DNA was found, however amount of DNA was not enough to be analyzed. § After DNA extraction, the scientist want to study a specific part of a gene to do sequencing. How scientist...

Polymerase chain reaction (PCR) BCH361- Practical 2 § In a crime scene, a sample of DNA was found, however amount of DNA was not enough to be analyzed. § After DNA extraction, the scientist want to study a specific part of a gene to do sequencing. How scientist solve these problem ? 3 § The solution is to do amplification of parts of DNA!! § Mainly there are two methods: Amplifying segment of DNA Polymerase chain reaction Cloning 4  PCR is a means to amplify a particular piece of DNA. è Amplify= making numerous copies of a segment of DNA. PCR can make billions of copies of a target sequence of DNA in short time. It is a laboratory version of DNA Replication in cells. è The laboratory version is commonly called “in vitro” since it occurs in a test tube while “in vivo” signifies occurring in a living cell. 5  So… è How the amplification will be done? è How you will determine your target sequence? è How the amplification will be specific for certain segment? You must to understand these questions 6  PCR does not copy all of the DNA in the sample. It copies only a very specific sequence of genetic code from a template DNA, targeted by PCR primers. It does require the knowledge of some DNA sequence information which flanks the fragment of DNA to be amplified (target DNA). Primer Primer 7  From this information two synthetic oligonucleotide primers may be chemically synthesised each complementary to a stretch of DNA to the 3’ side of the target DNA, one oligonucleotide for each of the two DNA strands (DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group). 8 Forward strand In a PCR reaction you need two primers to amplify the target sequence: èOne called: Forward primer, which have the same sequence of forward DNA strand and bind to the complementary reverse strand. èThe second called: Reverse primer, which have the same sequence of reverse DNA strand and bind to the complementary forward strand. Reverse strand *If there is only one primer, only one strand of the double stranded DNA will be amplified in the PCR reaction. 9  MgCl2 Additional reagents may included 10  PCR proceeds in THREE distinct steps Governed by Temperature: The double-stranded The reaction is rapidly The reaction is heated template DNA is cooled to an annealing to a temperature, denatured by heating, temperature to allow typically 72°C for typically to 95°C, to the oligonucleotide efficient DNA synthesis separate the double primers to hybridize to by the thermostable stranded DNA. the template. DNA polymerase. Denaturation: **Annealing: Extension: (95⁰C) (50-65⁰C) (72⁰C) 11  The double-stranded template DNA is denatured by heating, typically to 95°C, to separate the double stranded DNA (why?). Breaking the _______ bonds. Step 1 (94–97 °C ) 12  The reaction is rapidly cooled to the primer annealing temperature (50-65 °C) to allow the oligonucleotide primers to hybridize to single stranded template. Primer will anneal only to sequences that are complementary to them (target sequence). What is the type of the bond? Step 2 (50–65 °C ) 13  The reaction is heated to a temperature depends on the DNA polymerase used. Commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template Step 3 (72 °C ) 14 One cycle 15  At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons). In only 20 cycles, PCR can product about a million (220) copies of the target. 16  Post-PCR Identification analysis results Start your PCR the location of Primer design PCR using agarose PCR and visualize the target and primer optimization gel troubleshooting the results by sequence in the specificity electrophoresis AGE DNA template (AGE) 17  You want to study a mutation in a DLG3 gene and how it relate to memory: 1. Find the sequence of the gene from any website, eg.Ensebmle. 2. Determine your target region. The segment that you want to amplified is in the red square 5’ 3’ 3’ 5’ 3. Design the primers using primer design tool, eg.Primer3, then send them to any company who will synthesize them. 4. Make sure that the area that you want to study is between the primers (the region to be studied should be between the forward and reverse primer). 5. Check primer specificity by BLAST. 6. Optimize your PCR and trouble shooting. 18 7. Start PCR. 1. Denaturation: 95 °C 5’ 3’ 3’ 5’ 19 2. Annealing: 58 °C 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Forward primer: 5’ 3’ Reverse primer: 3’ 5’ 20 3. Extension: 72 °C 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Taq DNA polymerase 21 3. Extension: 72 °C 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 5’ Cycle # 1: 1 DNA amplified to 2 DNA 22 Cycle 2 1. Denaturation 2. Annealing 23  Cycle 2 3. Extension 3’ 3’ 3’ 3’ Cycle 3 3’ 3’ 3’ 3’ Target sequence After 30 cycles: Appeared after three cycles and 230 copy of target DNA !! start to accumulate 24 It is very important to know your product size è Our target sequence size is 350 bp 25 Marker Target sequence 26  Simplicity, easier methodology, sensitive, extensively validated standard operating procedure and availability of reagents and equipment Ø Genotyping. Ø RT-PCR. Ø Cloning. Ø Mutation detection. Ø Sequencing. Ø Microarrays. Ø Forensics. Ø Paternity testing. 27 Primer sequence Primer length GC% GC clamp Melting temperature (Tm) Annealing temperature (Ta) 29 1. Primer sequence: Must be complementary to flanking sequences of target region. Avoid: èComplementary sequences between primers. èRepeat (ex: ATATATAT) è misprime. è Runs (ex: AGCGGGGGAT) misprime. 2. Primer length: It is generally accepted that the optimal length of primers is 18-25 bp. Not too long nor too short 30 3. GC content: GC% = Number of G's and C's in the primer as a percentage of the total bases. Should be 40-60%. 4. GC clamp: Presence of G or C bases within the last five bases from the 3' end of primers. Not more than 2 G's or C's. 31 5. Melting temperature (Tm): What is Tm? Melting temperatures in the range of 50-60 °C generally produce the best results. Maximum difference between primer pairs is 5°C. The Tm of the primer can be calculated by the following formula: Tm = [(G + C) x 4] + [(A + T) x 2] 6. Annealing Temperature (Ta): The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Depends directly on length and GC composition of the primers. Too high Ta è produce insufficient primer-template hybridization. Too low Ta è lead to non-specific products caused by a high number of base pair mismatches. 32  How the amplification will be done? How you will determine your target sequence? How the amplification will be specific for certain segment? What are the requirements to carry PCR? 33