Chapter 8: Genetic Analysis and Recombinant DNA Technology

intrinsic properties of DNA

DNA strands separate when exposed to temperatures just below boiling

Restriction Endonucleases

enzymes able to clip DNA crosswise at selected positions/specific locations; able to recognize foreign DNA to act upon; protect bacteria and archaea against incompatible DNA of bacteriophage or plasmids

palindromes

sequences of DNA that are identical when read from the 5 prime to 3 prime direction on one strand and the 5 prime to 3 prime direction on the other strand

sticky ends

staggered symmetrical cuts that leave short tails; base pair with complementary tails on either DNA fragments or plasmids

restriction fragments

pieces of DNA produced by restriction endonucleases

ligase

necessary to seal sticky ends together by rejoining the phosphate-sugar bonds cut by endonucleases

reverse transcriptase

able to convert RNA into DNA

cDNA: complementary DNA

made from messenger, transfer, ribosomal, and other forms of RNA; useful in synthesizing eukaryotic genes from mRNA transcripts - free of introns

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats; short lengths of DNA with repeating nucleotides found in bacteria and archaea

Enzyme Cas9

recognizes and cuts out foreign DNA left behind by invading bacteriophages or plasmids in CRISPR; used and manipulated by scientists in cutting DNA

Polymerase Chain Reaction

rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques; mimics replication - can replicate DNA from a few copies to billions of copies in a few hours

primers

synthetic oligonucleotides of a known sequence of 15-30 bases that initiate the start of DNA amplification in polymerase chain reaction

DNA polymerases

enzymes responsible for the replication of DNA

PCR Technique

utilizes a thermal cycler that automatically performs cyclic temperature changes; three steps include denaturation (opening up or heating), priming (finding), and extension (copies or cooling); cyclic repetition of these steps amplifies the DNA

recombinant DNA technology

deliberately removing genetic material from one organism and combining with that of a different organism - genetic clones

cloning

removal of a selected gene from an animal, plant, or microbe where a gene is inserted into a vector that will be inserted into a cloning host and then a gene is translated into the protein product for which it codes

genomic libraries

collections of DNA clones that represent the entire genome of numerous organisms

cloning vectors

plasmids and bacteriopages

plasmids

con be transferred into appropriate host cells through transformation

bacteriophage

have the natural ability to inject DNA into bacterial hosts through transduction

gene cloning ingredients

gene of interest and cloning vector

synthetic biology

the creation of new biological molecules and organisms from scratch; self replicating bacterium from 4 nucleotides of DNA

gel electrophoresis

produces a readable pattern of DNA fragments; samples of DNA are placed in a soft agar gel and subjected to an electrical current. the negative charge on the phosphate groups cause the DNA to move toward the positive pole on the gel. the rate of movement of DNA through the gel is based on the size of the fragments. positions of DNA fragments are determined by staining them in the gel

analysis of DNA can

reveal genetic abnormalities and ancestry, determine if one sample of DNA is the same as another sample

hybridize

nucleic acids can bind together at their complementary bases

gene probes

a short stretch of DNA of a known sequence that will base pair with a stretch of DNA with a complementary sequence if one exists in the sample

Fluorescent in Situ Hybridization

used to identify genes on chromosomes and in identifying bacteria living in natural habitats without culturing them(very effective); detects RNA in cells and tissues. observed microscopically

DNA profiling (DNA fingerprinting)

a tool of forensic science that relies on the fact that DNA in individuals from the same species contains small differences; restriction enzymes cut the DNA in different places resulting in fragments of different lengths in the digested samples of different individuals

Restriction Fragment Length Polymorphism

restriction enzymes cut DNA at a specific recognition site; each of these two samples is digested with restriction enzymes and separated on an electrophoretic gel

sequence maps

the most detailed maps of a genome; give exact order of bases in a plasmid, chromosome, or genome

contigs

a larger, contiguous set of nucleotide sequences produced by the automated process in a computer program; put in the proper order to determine the entire sequence of DNA

editing

a human examines the sequence, looking for irregularities, frameshifts, and ambiguities

High-Throughput Genome Sequencing

sophisticated computers and systems developed to sequence genomes in a shorter period of time with less human effort

genomics and bioinformatics

manage the data produced through high-throughput screening by analyzing and classifying genes, determining protein sequences, and determining the function of the genes

annotating genomes

determining the functional information of a gene sequence

Single Nucleotide Polymorphism

only a single nucleotide is altered - important in individual traits

DNA Microarray Analysis

allows biologists to view the expression of genes in any given cell - tracks the expression of thousands of genes at once

Microarray Uses

developing sensitive diagnostic tests that search for a specific pattern of gene expression

interferons

peptides used to treat some types of cancer, multiple sclerosis, and viral infections such as hepatitis and general warts

interleukins

types of cytokines that regulate the immune function of white blood cells, used in cancer treatment

tumor necrosis factor

used to treat cancer

Remicade and Humira

biologics used to treat autoimmune disorders such as rheumatoid arthritis and Crohn’s disease

erythropoeitin

a peptide that stimulates bone marrow and used to treat some forms of anemia

human growth hormone

stimulates growth in children with dwarfism and prevents wasting syndrome

rh DNA se

a treatment that can break down the thick lung secretions of cystic fibrosis

tissue plasminogen activating factor

can dissolve potentially dangerous blood clots

PEG-SOD

a form of superoxide dismutase that minimizes damage to brain and other tissues after surgery or severe trauma

factor VIII

needed as a replacement blood-clotting factor in type A hemophilia

genetically modified organisms

recombinant organisms produced through the introduction of foreign genes

gene therapy

repair or correction of a faulty gene in humans suffering from fatal or debilitating disease

CAR-T cell treatment of cancer

modifies T cells that bind to cancer cells

CRISPR-Cas9 System

naturally occurring system in bacteria also known as gene drive