Molecular Biology - Mutation Detection Lecture PDF

MLS 420 - LECTURE | MOLECULAR BIOLOGY - LECTURE F2: Mutation Detection Professor: Dr. Milliem Ruzzlee Reyes Date: April 29, 2024 LEARNING POINTS...

MLS 420 - LECTURE | MOLECULAR BIOLOGY - LECTURE F2: Mutation Detection Professor: Dr. Milliem Ruzzlee Reyes Date: April 29, 2024 LEARNING POINTS Silent/Synonymous Mutation Missense, conservative & nonconservative Mutations Review mutation Known vs Unknown Mutations Detection of Known Mutations Detection of Unknown Mutations Review: Mutations Silent/synonymous mutation Base Insertion Base Substitution Missense, conservative & nonconservative mutation Together, they’re called base indels. Nonsense Mutation Frameshift Mutation Base substitutions can be Transition (Pyrimidine → Pyrimidine; Purine → Purine) ○ Cytosine-Thymine Transversion (Pyrimidine → Purine) ○ Adenine-Cytosine Nonsense mutation Happens during a strand slippage @mlstranses | 1  Detection of the Known Allele-Specific Oligo- nucleotide Hybridization ALSO KNOWN AS: “Dot-Blot” Method Alleles of a gene → Alternatives/variants of a gene due to mutations in the same place on a chromosome ○ Relies on specific binding of known chromogenic probes on DNA and detection of probes thereafter The probes that we have can either be designed to TARGET THE NORMAL SEQUENCE Because we already know the sequence that was mutated, we can probe the mutated sequence If we have a probe and finds its target DNA → hybridizes → chromogenic molecule will create color ○ Cannot find target sequence → No color during hybridization CYSTIC FIBROSIS CFTR (Cystic fibrosis transmembrane conductance regulator) is mutated Specific mutation is a deletion in delta 508 Known vs Unknown Mutations position (AGA → ATG) Known Mutations Unknown Mutations Create a probe for the normal and mutated sequence “I know there is a change!” “I know there is change!” Apply an example for the family “I know it’s from a mutation!” “I know there is a mutation!” Mother and father are Heterozygous = normal gene “I know its consequences!” “I may not know its consequences!” The child only has the mutated gene (Has cystic fibrosis) “I know what to look for!” “I don’t know what look for!” ○ Second child is normal, third child is heterozygous “I know where to look!” “I don’t know where to look!” @mlstranses | 2  ADD THE MIX (Sample 1 If we have a probe for the normal and probe for mutant, it will appear like and Sample 2) TO EACH the image above on the dot-blot OF THE WELLS, Probe found the normal and mutated gene because the parents have It will only fluorescence if the both. probe finds its ○ Sick child’s dot blot has an intense color compared to the other complementary sequence offspring Well (Left Description What Color to Right) Fluoresced…? Well 1 Gene A Normal NONE GRAY Well 2 Gene A Mutant Sample 2 RED Well 3 Gene B Normal Sample 1 and 2 YELLOW Well 4 Gene B Mutant NONE GRAY DNA We can use this for known Microarray mutations (probes are involved) Well 5 Gene C Normal NONE GRAY Probes are present in the array Sample 1 is labeled with a Well 6 Gene C Mutant Sample 1 GREEN green, fluorescent molecule Sample 2 is a red molecule ○ Together in one mix DNA Even more specific ○ In the microarray, each of Sequencing How? Get the sample that we want and sequence DNA → the wells contain a probe compare DNA to standard/normal DNA for a different portion of MAXAM GILBERT the DNA/ genes ○ Any sequencing method will do as long as you Well 1 → Gene A Normal compare the sequences Well 2 → Gene A Mutant ○ Read from BOTTOM TO TOP Well 3 → Gene B Normal Well 4 → Gene B Mutant, etc. @mlstranses | 3  1. LEFT PROBE will have a fluorophore → detects signals, same with all the probes a. Forward primer and half of the probe sequence 2. RIGHT PROBE will have the other half of the probe sequence and a reverse primer. 3. For all the 50 pairs (of probes) → What is unique to each is the probe sequences (What gene to look out for) and the sizing (differentiate the Automated (Dye terminator; dye primer) sequences) since they have the same fluorescent signal ○ Result = electropherogram 4. Incubate 2 probes with denatured DNA so they can hybridize a. If they find their perfect match (½ and other half) ⇒ PERFECT Multiplex Many PCR methods can be used in detecting mutations PAIRING Ligation MLDPA makes use of multiplex PCR with different probes for different Dependent b. If mutation is present, it won’t be straight ⚠️ deletions and duplications in up to 50 different genomic DNA/RNA sequences 5. Add DNA ligase, it will only ligate those PERFECTLY PAIRED (without Probe ○ Cannot detect base substitutions, only base indels mutation) Amplification (MLDPA) a. The ones without a mutation b. Anything that has a mutation will not be straight(those with kinks) i. Will not form one long strand – JUST the left and right For PCR you need a forward and reverse primer– if it was not ligated together, the primers are located (ligated) differently and is not feasible for PCR If you have no mutations, they are ligated together (left and right) you will have an amplicon because you have a ligation No ligation = no amplicons WHAT TO DO NEXT IN MLDPA? HOW DOES IT WORK? You have 2 probes per gene you are looking for (up to 50) Software will first reference your test sample to the normal 50 left & 50 right probes; they pair together In the normal, everything is amplified (reference) because there is no mutation ○ The test sample should have equal peaks ○ Unequal peaks = mutation because some were not amplified @mlstranses | 4  Arrange those different amplicons by their position in the chromosome ‘ SAMPLE DESCRIPTION RESULT X No change in ratio from One NORMAL Y Ratio decreased HETEROZYGOUS DELETION ○ Insertion = more than the normal copy number (1) Detection of the Unknown Single Strand Conformation Polymorphism (SSCP) Takes advantage of the secondary structures of ssDNA will have different foldings in non-denaturing conditions ○ Different foldings will have different mobilities in the electrophoretic gels ○ In AGE, we want DNA samples to be denatured so they migrate according to size Different folding patterns = Different migration pattern For a specific sequence, there is a specific secondary structure that it will conform to For example in the WILD TYPE, you denatured DNA and through rapid cooling you allow it to fold by itself In the mutant, it can only fold into shape DIFFERENT from the wild type ○ Migration will be different in the gel [show migration patterns] In the diploid gene, you have two sets of alleles (normal copies) Remember to always compare with the normal/wild type – if they look alike, ○ The ratio of the normal copy is 1 there’s no mutation ○ The reference copy, normal = 1 copy number since they are equal ○ If there are changes in appearance - there is mutation SSCP has the limitation → Fragment size limit 150 - 200 bp ○ Detects 80-90% unknown mutations because of the limitation it has @mlstranses | 5 Denaturing Gel Modification of AGE that uses different intensities of a denaturing Gradient environment in the same setup Electrophoresis Denaturing Chemicals ○ Formamide and Urea (added to the loading buffer) Fragments melt in a step-wise manner based on its melting profile How to make a denaturing gradient? In the normal preparation of gel, the powder will be added with loading buffer and added to formamide to make it more denaturing → POUR and wait to solidify In gradient gel, prepare a low-concentration solution of [example] urea and molten gel ○ Then high concentration of urea and gel ○ Attach to a regulator pump that will be placed in the casting tray ○ In the beginning, High concentration will be prioritized first then gradually replaced by low concentration as we go up At the very top of the gel = lowest denaturing conditions Bottom = highest denaturing conditions(urea/formamide content) Orientation of the gel = vertical → POLYACRYLAMIDE GEL (Vertically casted) DNA MELTING PROFILE DNA will denature in small pockets /step-wise manner First to denature are the areas with the A-T content (weakest bonds) ○ Next, second to the highest AT content ○ Last will be the highest G-C content (strongest bonds) How does it translate to the gel? @mlstranses | 6  Heteroduplex ○ We always have the reference wildtype Analysis ○ 2nd lane has a mutation, and ALWAYS COMPARE WITH THE Homoduplex Heteroduplex WILDTYPE Instead of getting to know what the mutation is, we have an idea Perfectly paired dsDNA dsDNA with one or more ○ Wt-Wt mismatched pairs ○ Faster denaturation = more A-T content → MUTANT 1 (Wildtype-Wildtype) ○ Wt-Mu (Wildtype-Mutant) ○ Even higher A-T content if higher placement on the lane → ○ Mu-Mu (Mutant-Mutant) Reduced mobility in gel MUTANT 2 Faster mobility in gel that results in a bulge ○ What if lower than wild type? → Much higher G-C content Problem: Difficult to replicate afterward ○ Solution: Use temperature as the denaturant instead of chemicals Looking for heteroduplexes - because they cannot be found if there are no mutations IF BASE INDELS … → bulge IS stable, and can be seen under electron microscope → CAN BE USED for the detection IF BASE SUBSTITUTION… → bulge cannot be retained EASILY and not easily detected Temperature Modification of DGGE that uses temperature instead of a denaturing Gradient Gel chemical Electrophoresis More reproducible and reliable than chemical gradient Restriction Uses diff restriction cut sites to detect mutations Fragment Same principle as DNA Fingerprinting Length Mutations =Change in cut sites = Different fragments Polymorphism WEAKNESS: Detected mutations are those that affect palindromic cut sites it is involved in Another method of looking for heteroduplexes (analyze the picture) ○ Loss of cut site = recognized by them = fragment lines change Heteroduplex sample → harder time passing through the gel, RETAINED ○ Anywhere other than palindromic site = cannot be detected FARTHER ON Cut with different restriction enzymes Homoduplex stays at the LOWER PORTION → MUTANT @mlstranses | 7  SINGLE STRAND SPECIFIC NUCLEASES In some cases, the wildtype and the homoduplex have the same ❗ migration IGNORES HOMODUPLEXES COMPLETELY Sees a heteroduplex - cut the bulge and release the fragment ○ AFTERWARDS, run it in the gel Whatever lane has bands, it will have the mutation → HETERODUPLEX The rest will not have a band → NORMAL @mlstranses | 8