Pr Lecture IVa_Gene Amplification and PCR_Lecture.pptx

Gene Amplification Gene Amplification Gene amplification refers to an increase in the copy of a particular gene. Here, the number of copies of a gene increases without an increase in the number of copies of other genes. There may also be an increase in the gene products, i.e. RNA and protein. It h...

Gene Amplification Gene Amplification Gene amplification refers to an increase in the copy of a particular gene. Here, the number of copies of a gene increases without an increase in the number of copies of other genes. There may also be an increase in the gene products, i.e. RNA and protein. It happens naturally as well as done artificially to get the desired gene product. E.g. Multiple copies of a gene are produced in cancer cells sometimes, it occurs naturally on receiving signals from a cell or environment. Gene amplification is artificially done in a lab by polymerase chain reaction (PCR). The piece of DNA or the gene that gets amplified naturally or artificially is known as an amplicon. Natural Gene Amplification The DNA is duplicated inside the cell by replication. Replication does not change the number of genes present in a cell under normal circumstances. A gene amplification naturally can occur by gene duplication. There are various ways by which gene duplication can occur naturally. •Replication and repair slippage – An error in replication and repair leads to mutation. •Ectopic recombination – Crossing-over between non-homologous chromosomes. It leads to chromosomal rearrangement. •Aneuploidy and Polyploidy – Aneuploidy refers to the loss or gain of one or more chromosomes in a cell, e.g. a human cell with 45 or 47 chromosomes. Polyploidy refers to an increase in the whole set of chromosomes in a cell, e.g. triploid, hexaploid, etc. •Retrotransposition – They are a type of transposons that copy-paste themselves at different locations through RNA intermediates. Many tumour cells show overexpression of the amplified gene. Amplification is useful for diagnosing disease and understanding its progression. It is also a cause of developing drug resistance in cancer cells. Artificial Gene Amplification Artificial gene amplification is done for research, diagnosis, recombinant DNA technology and to get the desired gene product. Various ways by which gene amplification is done artificially are as follows: •Polymerase chain reaction (PCR) – Polymerase chain reaction or PCR is commonly used to produce multiple copies of the desired gene in the lab. It requires a thermostable polymerase. The steps of polymerase chain reaction are denaturation, primer annealing and extension of primers. It is a common method for gene amplification in modern science and biotechnology. This technique is used in labs to make billions of copies of the desired gene for various purposes such as research, diagnostic and therapeutic use. •Ligase chain reaction (LCR) – Ligase chain reaction or LCR is also a method of gene amplification. It requires a thermostable ligase as well as a polymerase. Two probes are ligated using DNA ligase, which is then amplified by PCR. It is more specific as compared to PCR. It is widely used to ascertain point mutation such as in genetic diseases. •Transcription-mediated amplification (TMA) – This technique utilises RNA polymerase and the enzyme reverse transcriptase. Here, RNA amplicon is produced. It involves RNA transcription by RNA polymerase and DNA synthesis by reverse transcriptase. It rapidly amplifies the target gene (RNA/DNA). Multiple pathogenic organisms can be detected simultaneously in a single tube by this method. Polymerase chain reaction (PCR) Polymerase chain reaction (PCR) 7  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 ? 8  The solution is to do amplification of parts DNA!!  Mainly there are two methods: of Amplifying segment of DNA Polymerase chain reaction Cloning 9 • PCR is a means to amplify a particular piece of DNA .  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.  The laboratory version is commonly called “in vitro” since it occurs in a test tube while “in vivo” signifies occurring in a living cell. 10 • So…  How the amplification will be done?  How you will determine your target sequence?  How the amplification will be specific for certain segment? You must to understand these questions 11 • 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 12 • 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). 13 Forward strand • In a PCR reaction you need two primers to amplify the target sequence: One called: Forward primer, which have the same sequence of forward DNA strand and bind to the complementary reverse strand. 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. 14 MgC l2 Additional reagents may included 15 • PCR proceeds in THREE distinct steps Governed by Temperature: • The double-stranded template DNA is denatured by heating, typically to 95°C, to separate the double stranded DNA. Denaturation: (95⁰C) • The reaction is rapidly cooled to an annealing temperature to allow the oligonucleotide primers to hybridize to the template. **Annealing: (50-65⁰C) • The reaction is heated to a temperature, typically 72°C for efficient DNA synthesis by the thermostable DNA polymerase. Extension: (72⁰C) 16 Mechanism of PCR • 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 ) 18 • 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 ) 19 • The reaction is heated to a temperature depends on the DNA polymerase used. • Commonly a temperature of 72°C is used with this enzyme. • This means that 72°C is the optimum ………………… of DNA polymerase. • At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template Step 3 (72 °C ) 20 One X 25 cycle 21 • 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. 22 Number of copies: 2n Where, n= no. of cycles Identification the location of the target sequence in the DNA template Primer design and primer specificity PCR optimization Post-PCR analysis results using agarose gel electrophoresis (AGE) PCR troubleshooting Start your PCR and visualize the results by AGE 24 • 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. 7. Start PCR. 18 1. Denaturation: 95 °C 5’ 3’ 3’ 5’ 19 2. Annealing: 58 °C 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ Forward primer: 5’ 3’ 5’ Reverse primer: 3’ 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’ Cycle # 1: 1 DNA amplified to 2 DNA 5’ 22 2 1. Denaturation 2. Annealing 23 2 3. Extension 3’ 3’ 3’ 3’ Cycle 3 3’ 3’ 3’ 3’ Target sequence Appeared after three cycles and start to accumulate After 30 cycles: 230 copy of target DNA !! 24 How you will make sure that you target sequence is amplified? It is very important to know your product size, why?  Our target sequence size is 350 bp 32 Marker Target sequence 33 • Simplicity, easier methodology, sensitive, extensively validated standard operating procedure and availability of reagents and equipment  Genotyping.  RT-PCR.  Cloning.  Mutation detection.  Sequencing.  Microarrays.  Forensics.  Paternity testing. 34 Primer sequence Primer length GC% GC clamp Melting temperature (Tm) Annealing temperature (Ta) 35 1. Primer sequence: • Must be complementary to flanking sequences of target region. • Avoid: Complementary sequences between primers. Repeat (ex: ATATATAT)  misprime. Runs (ex: AGCGGGGGAT)  misprime. Mismatch at 3’ end. Cross Homology. 2. Primer length: • It is generally accepted that the optimal length of primers is 18-25 bp. • Not too long nor too short 36 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 . 37 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  produce insufficient primer-template hybridization. • Too low Ta  lead to non-specific products caused by a high number of base pair mismatches. 38