L5 Restriction enzymes and dna fingerprinting

• gel electrophoresis

DNA fragments are separated by size when passed through an agarose or polyacrylamide gel under an electric field. Smaller fragments migrate farther.

Southern and Northern Blots:

• Southern Blot: Used to detect specific DNA sequences in a sample by transferring DNA to a membrane and hybridizing with a complementary labeled probe.

• Northern Blot: Similar to Southern but used for RNA detection.

Restriction Mapping:

Creates a map of restriction sites within DNA by cutting with restriction enzymes and analyzing fragment sizes via gel electrophoresis.

Paternity Testing & DNA Fingerprinting:

• Relies on restriction fragment length polymorphisms (RFLPs) and variable number tandem repeats (VNTRs) to determine genetic relationships or identify individuals.

Restriction Enzymes and Sites:

• Enzymes recognize specific palindromic sequences and cleave DNA, generating either blunt or sticky ends.

Blunt Ends and Sticky Ends:

• Sticky ends are single-stranded overhangs; blunt ends lack overhangs.

• Sticky ends facilitate the annealing of complementary DNA fragments.

Plasmids and Hybrid Plasmids:

• Plasmids are small, circular DNA molecules used in cloning. Hybrid plasmids carry foreign DNA inserted using ligation.

Restriction Fragment Length Polymorphisms (RFLPs):

• Variations in DNA fragment sizes between individuals, useful for gene mapping and forensic analysis.

Pulse-Field Gel Electrophoresis:

• Allows separation of very large DNA molecules by applying an alternating electric field.

Transfection & Transgenics:

• Transfection: Introduction of DNA into eukaryotic cells.

• Transgenics: Organisms genetically modified to carry foreign DNA.

Electroporation:

• Uses an electric field to create temporary pores in cell membranes to introduce DNA.

Protein Tags & GFP:

• Protein Tags: Used for tracking or purification of proteins.

• GFP (Green Fluorescent Protein): Reporter protein for monitoring gene expression or protein localization.

Restriction Enzymes:

• Cut DNA at specific sequences.

• Essential for molecular cloning and RFLP analysis.

Microarrays

Microarrays are used to analyze gene expression. A grid of DNA probes on a solid surface binds to complementary DNA or RNA from a sample. By detecting hybridization, the expression levels of thousands of genes can be measured simultaneously.

Immunoblotting

Immunoblotting (Western blotting) detects specific proteins in a sample. Proteins are separated via gel electrophoresis, transferred to a membrane, and probed with antibodies that recognize the target protein.

Molecular Cloning​

• Involves isolating and copying specific DNA sequences.

• A foreign DNA piece is inserted into a vehicle (vector), such as a plasmid or phage.

• The vector is introduced into a host cell, which replicates the DNA through cellular replication.

• Techniques include gene cloning, recombinant DNA technology, and genetic engineering.

Shotgun Cloning

A method to create genomic libraries by randomly fragmenting DNA and cloning these fragments into vectors. This approach helps in sequencing entire genomes.

PCR (Polymerase Chain Reaction)

PCR amplifies specific DNA sequences using cycles of denaturation, annealing of primers, and extension by DNA polymerase. It’s widely used in diagnostics, cloning, and forensic science.

DNA Sequencing

• The process of determining the nucleotide sequence of DNA. Modern techniques include Sanger sequencing and next-generation sequencing.

DNA Cloning​

• Involves isolating a DNA fragment and inserting it into a vector for replication.

• The host organism replicates the DNA alongside its own DNA, allowing the cloned DNA to increase in copy number.

Restriction Sites

Specific sequences of DNA where restriction enzymes cut. They are like "scissors" that recognize specific patterns in DNA and make a precise cut.

Blunt Ends and Sticky Ends

• Blunt Ends: DNA is cut straight across, leaving no overhangs.

• Sticky Ends: Cuts leave single-stranded overhangs, which can easily join with complementary DNA strands, like puzzle pieces.

Plasmids and Hybrid Plasmids

• Plasmids: Small circular DNA found in bacteria that replicate independently of the main chromosome.

• Hybrid Plasmids: Plasmids with a piece of foreign DNA inserted. They are used to clone or express genes in molecular biology.

Restriction Fragment Length Polymorphisms (RFLPs)

• Differences in DNA fragment sizes when cut by restriction enzymes. These differences can be used for genetic fingerprinting or paternity tests.

Yac’s SV40

• YACs (Yeast Artificial Chromosomes): Tools used to clone large pieces of DNA in yeast cells.

• SV40: A virus often used in molecular biology as a vector to deliver genetic material into cells.

Bacteriophages

• Viruses that infect bacteria. Scientists use them as tools to deliver DNA into bacteria for experiments.

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Cosmids

• A hybrid between plasmids and bacteriophages, designed to carry larger DNA fragments. Useful for cloning larger genes.

cDNA and Reverse Transcriptase

• cDNA: "Complementary DNA" synthesized from RNA. Represents only the genes being actively expressed.

• Reverse Transcriptase: An enzyme that converts RNA into DNA. It’s used to create cDNA from mRNA.

• liposomes delivery

• Liposomes are tiny fat bubbles used to deliver DNA or drugs into cells. They merge with the cell membrane to release their contents inside.

Biolistic Delivery

• A method of introducing DNA into cells using a "gene gun." Tiny metal particles coated with DNA are shot into cells.

Reading Frames

DNA is read in groups of three bases (codons). The reading frame determines which codons are read to make proteins. Shifting the frame can change the entire protein sequence.

• Site-Directed Mutagenesis

A technique to introduce specific changes into a DNA sequence, like editing a single "letter" in the genetic code.

Yeast Two- and Three-Hybrid Analysis

Methods to study how proteins interact:

• Two-Hybrid: Tests if two proteins bind to each other.

• Three-Hybrid: Adds a third molecule (like RNA) to see if it mediates interactions between two proteins.