Molecular Genotyping - MCB 104 Flashcards
Allows you to find what genotype is responsible for a particular phenotype. specifically used to identify what genotype is responsible for a human disease.
Why do we want to do molecular genotyping?
If a person is diagnosed with a genetic disease, knowledge of the genotype that caused the disease could save his/her life. And the chances of her/him passing the disease onto the off spring can be known.
What is the first thing a geneticist may ask before they begin molecular genotyping?
Do the symptoms (phenotypes) align with a known/ already documented disease? If yes, then do we know the mutation that causes this disease?
If a person is diagnosed with a disease, what types of genotypes can the person have and how can they know which of the three might explain the disease?
AA - Homozygous Aa - Heterozygous aa - Recessive In this scenario scientists should already know the disease and what type of genotype is responsible for this disease. That way after genotyping, they know exactly what to look for to confirm their diagnosis. BUT SCIENTISTS DO NOT ALWAYS KNOW WHAT MUTATION CAUSES THE DISEASE.
But how do scientists identify the disease causing genotype/allele?
1) First you isolate the gene from the individual’s genome 2) then you look for the alleles to see whether the individual is homozygous or heterozygous?
Scientists identify the disease causing allele by isolating the gene from the individual’s genome. BUT How do you isolate your target gene? because genes (protein coding regions) only make up a fraction of an entire human genome. this is like finding a needle in the haystack?
By using PCR - Polymerase Chain Reaction
What is the PCR strategy though?
Process of amplifying a short sequence in that DNA [the gene sequence because remember you already know where to look you are just trying to confirm the allelic forms now] that may have different allelic forms to help identify the disease causing allele.
Why do we need to amplify a short sequences in the DNA if we already have the sequence?
Because the segment is such a small piece that scientists need to make billions of copies to work with. To make billions of copies is what amplification is.
Ok then what does PCR need?
1) a test tube 2) few reagents 3) heat source.
Explain the steps involved in PCR
1) extract a portion of DNA [this portion is the sequence that contains the gene sequence too] from a cell. 2) add the DNA to a PCR test tube. 3) add primer 1 to the test tube so it can attach to 5’ end 4) add primer 2 to the test tube so it can attach to 3’ end 5) add nucleotides to the test tube 6) add DNA polymerase to the PCR tube. 7) place the test tube in thermal cycler so it can apply heat and cool at specific times which is an important part of making the reaction work. First Cycle begins: 8) thermal cycler heats up to 95 degrees, dsDNA strands separate 9) then thermal cycler cools down, and the primers quickly crowd the area between separated strands, and the complementary strands attach. 10) then temperature goes up again and RNA polymerase binds to the primer and begins to add nucleotides until it reaches the end of the strand and then falls off generating a hybrid strand. Second cycle begins: steps 8, 9, 10 are repeated, this time with the hybrid strand. Third cycle begins: During this cycle the desired products begin to appear = 2. cycle 4: 8 products will appear. cycle 30: there are over a billion fragments that contain only our target sequence and only ~60 copies of longer length DNA.
special property of PCR test tube?
PCR test tube that is designed to take manage tons of even heating and cooling
What is a primer and what is their purpose? How do primers know what part of the DNA to attach to?
Primers are short pieces of DNA that are made in a laboratory to copy the DNA target sequence and make many copies. [~17-26 nucleotides long] In a PCR experiment, two primers are designed to match to the segment of DNA you want to copy (or amplify). Through complementary base pairing, one primer attaches to the top strand at one end of the segment of interest [3’ end], and the other primer attaches to the bottom strand at the other end [5’ end]. Because primers are made in the laboratory, scientists specified the sequence before hand and primers rarely make a mistake in identifying what sequence to attach to.
Why do we add nucleotides to the PCR test tube?
these are used to create billions of copies of the DNA.
Why do we add DNA Polymerase? What is a special property of DNA polymerase used in PCR?
DNA polymerase attaches matching nucleotides to one another. This DNA Polymerase is designed to withstand high temperatures - it is from a bacteria that grows in hot springs.
PCR is used to amplify the target sequence. BUT how do we know it is amplifying? What are the end products that we want from PCR or the desired products?
Fragments with two strands that begin with primer 1 and end with primer 2. These are copies of the DNA that we amplified. At stage 3 only 2 fragments will appear, but as we subject the PCR to more cycles, more fragments will appear. by cycle 30 we have billions of copies and nearly purely target sequence fragment sample.
Ok so in card 4 we identified 2 steps to identifying the genotype. first was isolate the gene and amplify with PCR… that we just did .. second is to look at the alleles and see if they are homozygous or heterozygous. Or in other words analyze the gene sequence. How do you do that?
PCR products can be analyzed in two ways: 1) sequence them to find if the alleles are heterozygous or homozygous by Sanger sequencing 2) Using the difference in their size subject them to Gel electrophoresis to find the sequences that are abnormally short or long.
