15-Biotechnology

Recombinant DNA Technology

• Making Recombinant DNA Molecules

  • Creation involves cutting and joining DNA from different sources.

• Using Cloning and Expression Vectors

  • Vectors are tools that carry foreign genetic material into another cell.

• Ampicillin/X-gal Double-Selection Assay

  • Method for selecting transformed bacteria.

• Applications of Recombinant DNA Technology

  • Used in medicine, agriculture, and forensic science.

Forensic DNA Profiling

• RFLP and STR Loci

  • Use of various genetic markers to create individual DNA profiles.

• DNA Fingerprints

  • Unique patterns derived from analysis of specific DNA regions.

• Mitochondrial DNA Forensics

  • Utilizes mitochondrial DNA for identification, beneficial in cases with degraded samples.

Creating Recombinant DNA Molecules

• Digestion of DNA

  • Use of restriction enzymes like HindIII to cut DNA at specific sites.

• Combining DNA Fragments

  • Sticky ends of complementary fragments pair to form recombinant DNA.

• Role of Ligase

  • Seals nicks in the sugar-phosphate backbone of DNA strands.

Cloning Vectors and pUC18

• Function of a Cloning Vector

  • Used to replicate isolated genes in host cells.

• Components of pUC18 Cloning Vector

  • ampR: Ampicillin resistance gene.

  • lacZ+: Genecoding for beta-galactosidase enzyme.

  • Polylinker Region: Cluster of restriction sites for inserting foreign DNA.

• Insertion Consequence

  • Inserting foreign DNA disrupts lacZ+ and makes the plasmid lacZ-.

Using pUC18 as Cloning Vector

• Linearization Process

  • Plasmid cut by EcoRI to facilitate gene insertion.

• Mixing and Transformation

  • Resulting recombinant plasmid introduced into bacteria through transfection.

Antibiotic Selection in Cloning

• Antibiotic Use

  • Only bacteria with the ampR gene can survive in presence of ampicillin.

X-gal Selection for Cloned DNA

• Function of X-gal

  • Cleaved by b-galactosidase to produce blue dye for identifying positive clones.

• X-gal Assay Outcomes

  • Blue indicates lacZ+ (non-recombinant), white indicates lacZ- (recombinant).

Expression Vectors and Blue-White Assay

• Use of lacZ Gene

  • Disruption of lacZ gene by foreign DNA results in white colonies.

Summary of Selection Methods

• Cells and Plasmids

  • Surviving cells carry recombinant plasmids, indicated by color change in blue-white assay.

Agrobacterium Ti Plasmid for Plant Gene Transfer

• Transfer Mechanisms

  • TL and TR sequences facilitate the integration of foreign DNA in plant cells.

Transgenic Crops with Glyphosate Resistance

• EPSP Synthase Gene

  • Herbicide glyphosate targets this enzyme critical for amino acid biosynthesis.

• Integration Under Viral Promoter

  • Promoter in Ti plasmid enables high expression of EPSP synthase in transgenic plants.

Callus and Plant Regeneration

• Definition of Callus

  • A mass of undifferentiated cells that can regenerate complete plants.

Forensic DNA Profiling Methods

• RFLPs and STRs

  • Both methods generate unique DNA patterns for forensic identification.

• Electrophoretic Profiles

  • Different patterns correspond to the DNA fingerprints of individuals.

Short Tandem Repeats (STRs)

• Description and Analysis

  • STRs are variable repeat sequences; PCR can be used to analyze them quickly and efficiently.

• Example of STRs

  • D7S820 locus, contains variable numbers of GATA repeats.

FBI's CODIS STR Loci

• Core STR Markers

  • Database includes 13 essential STR markers.

• Unique Identifiers

  • Each locus displays variable number of repeats across individuals.

DNA Fingerprinting Techniques

• Identifying Suspects

  • Match DNA from crime scene with suspects; involves PCR analysis.

Amelogenin Test for Sex Identification

• Amelogenin Gene

  • Used for determining sex in DNA samples; longer in Y chromosome compared to X.

Example DNA Evidence in Historical Cases

• Romanov Family Identification

  • Mitochondrial DNA analysis to identify remains; Anna Anderson's case highlighted.