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GENETICS AND NEURO DISORDERS, Papers of Biology

DESCRIBES NEURO GENETIC DISEASES

Typology: Papers

2023/2024

Uploaded on 11/06/2024

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Name: MACDONALD CHIEMEZIE MBAGWU
Date: 15/04/2024
Course: MOLECULAR MEDICICINE AND MEDICAL GENETICS
ASSIGNMENT.
School: VICTORIA UNIVERSITY OF BARBADOS
A. POLYMERASE CHAIN REACTION
Overview:
● Goal: Amplify small quantities of DNA
● Clinical relevance in modern medicine and clinical genetics: Diagnostic tool (ex. HIV, herpes
encephalitis).
Process:
● Denaturation: Heat DNA to 95°C to separate strands
● Annealing: Cool sample to 55°C
- Add DNA primers
- Add heat-stable DNA polymerase
- Add deoxynucleotide triphosphates (dNTP)
- Taq polymerase: Heat-stable DNA polymerase
- DNA primers anneal to desired portion of DNA strand
- Every PCR uses 2 primers to flank the desired region of transcription
● Elongation: Increase temperature to 72°C
- DNA polymerase adds dNTPs to DNA primer
●This process is repeated many times → Amplify DNA
● PCR results can be analyzed via gel electrophoresis
Reverse Transcriptase PCR:
●mRNA → cDNA → PCR
● Reverse transcriptase enzyme added prior to PCR
A1. CRISPR/Cas9
Overview:
● Goal: Gene editing (deletions, mutations, or insertions)
● Use: Genetic manipulation, cancer research, viral research, genome study
Process:
● Cas9: Bacterial endonuclease used to locate and cleave host DNA
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Name: MACDONALD CHIEMEZIE MBAGWU

Date: 15/04/

Course: MOLECULAR MEDICICINE AND MEDICAL GENETICS

ASSIGNMENT.

School: VICTORIA UNIVERSITY OF BARBADOS

A. POLYMERASE CHAIN REACTION

Overview: ● Goal: Amplify small quantities of DNA ● Clinical relevance in modern medicine and clinical genetics: Diagnostic tool (ex. HIV, herpes encephalitis).

Process:

● Denaturation: Heat DNA to 95°C to separate strands ● Annealing: Cool sample to 55°C

  • Add DNA primers
  • Add heat-stable DNA polymerase
  • Add deoxynucleotide triphosphates (dNTP)
  • Taq polymerase: Heat-stable DNA polymerase
  • DNA primers anneal to desired portion of DNA strand
  • Every PCR uses 2 primers to flank the desired region of transcription ● Elongation: Increase temperature to 72°C
  • DNA polymerase adds dNTPs to DNA primer ●This process is repeated many times → Amplify DNA ● PCR results can be analyzed via gel electrophoresis Reverse Transcriptase PCR: ●mRNA → cDNA → PCR ● Reverse transcriptase enzyme added prior to PCR

A1. CRISPR/Cas

Overview: ● Goal: Gene editing (deletions, mutations, or insertions) ● Use: Genetic manipulation, cancer research, viral research, genome study Process: ● Cas9: Bacterial endonuclease used to locate and cleave host DNA

● Step 1: Cas9 locates a region of DNA complementary to its guide RNA ● Step 2: Cas9 makes single or double stranded breaks at target site ● Step 3A: Non-homologous end joining imperfectly repairs break → Mutation ● Step 3B: Donor DNA used to fill the gap via homologous recombination → Insertion

B. SOUTHERN, WESTERN, NORTHERN BLOT

Overview: ● Goal: Identify the amount of protein, DNA, or RNA present in a sample ● Clinical relevance in modern medicine and clinical genetics : Identifying size, identity, or quantity of DNA, RNA, or protein in a sample.

All techniques uses Gel Electrophoresis with different samples.

Gel Electrophoresis:

● Step 1: Heat and/or denature sample ● Step 2: Pipette sample into separate wells in agarose gel ● Step 3: Place gel in solution, run electric current through solution - DNA and RNA are negatively charged → Migrate to (+) electrode - Proteins coated with (-) charged substance → Migrate to (+) electrode ● Step 4: Transfer gel to filter paper ● Step 5: Incubate filter paper with Ab or radioactive probe which will bind to DNA/RNA/protein ● Step 6: Develop film of your filter paper, observe how far each sample migrated Southern, Western, Northern Blot Specific samples detected and peculiar technique: ● Southern Blot: Sample enzymatically cleaved prior to electrophoresis - Radiolabelled DNA probe marks sample for development - Detection of genes, specific nucleotide sequences ● Western Blot: Protein sample, antibody binds to protein for development. Detection of protein expression.

 Northern Blot: RNA sample

Detection of level of gene expression, mRNA  South western Blot: Detects DNA binding proteins.

C. Fluorescent In Situ Hybridization :

● Goal: Fluorescent DNA probe binds specific genetic regions of interest ● Clinical relevance in modern medicine and clinical genetics : Detect microdeletion, translocation, duplication - Useful when one region of DNA is in question ● Process: Karyotype affixed to slide → DNA/RNA probe added - Fluorescence detected