CH 10 Genetic Analysis and Genetic Engineering

What is applied science?

using applications from basic research science

Examples of applied science

• Utilizing DNA to identify a suspect in a crime

• Fixing the underlying genetic mutation to treat disease

• Utilizing RNA regulatory molecules to permanently “fix” diseases

Properties of DNA (3)

• Can be unwound by helicase

• Strands separate when exposed to temperatures below boiling

• Strands will regain their double-stranded form when the DNA is cooled

What are restriction endonucleases

• AKA restriction enzymes

• are responsible for cleaving DNA into fragments at or near specific recognition sites (restriction sites)

Properties of Restriction Endonucleases (6)

1. are enzymes

2. clip DNA crosswise at selected positions

3. recognizes foreign DNA

4. capable of breaking phosphodiester bonds between adjacent nucleotides

5. protects bacteria and archaea from bacteriophage or plasmids

6. recognizes a sequence of 4 to 10 base pairs

What are palindromes

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

What are sticky ends

• are staggered symmetrical cuts (done by restrictive enzymes) that leave short tails

• 4 to 5 bases on each strand

T/F: Restriction enzymes recognizes and cleaves DNA at the palindrome sequence

True

Restriction enzymes recognizes and cleaves DNA at the palindrome sequence. How? List the steps

1. A restriction enzyme recognizes and cleaves DNA at the site of a specific palindromic sequence

2. Cleavage can produce sticky ends that accept complementary tails for gene splicing

3. Sticky ends can be used to join DNA from different organisms by cutting it with the same restriction enzyme

What are restriction fragments?

pieces of DNA produced by restrictive enzymes

What are restriction fragment length polymorphisms (RFLPs)?

the differences in the cutting pattern of specific restrictive enzymes causes restrictive patterns of different lengths

T/F: RFLPs allows for direct comparison of DNA of two different organisms at a specific site

true

What is ligase

• an enzyme

• necessary to seal sticky ends together

What is the main application of ligase

final splicing of genes into plasmids and chromosomes

What is reverse transcriptase

• an enzyme responsible for converting RNA into DNA

• can replicate HIV and other retroviruses

What is cDNA and its function

• Complementary DNA

• synthesizes eukaryotic genes from mRNA transcripts

What is cDNA made from

• messenger RNA

• transfer RNA

• ribosomal RNA

• other forms of RNA

What are the steps of making cDNA from eukaryotic mRNA?

1. Transcription of eukaryotic DNA makes precursor mRNA

2. Splicing removes introns from precursor mRNA, making final mRNA for translation

3. mRNA is extracted from cells

4. Reverse transcriptase synthesizes DNA from mRNA

5. DNA polymerase completes cDNA (no introns)

What is the function of CRISPR

enzymes recognize and cuts out foreign DNA left behind by invading bacteriophages or plasmids

How do scientists use CRISPR

they exploit the system to cut DNA in just about any organism exactly where they want to

Applications of CRISPR

DNA Editing (find and replace function)

1. cell is transfected with an enzyme-complex containing: guide molecule, healthy DNA copy, and DNA-cutting enzyme

2. guide molecule finds target DNA strand

3. DNA-cutting enzyme cuts off target DNA strand

4. defective DNA strand is replaced with a healthy copy

What is the purpose of gel electrophoresis

to produce a readable pattern of DNA fragments

Process of using gel electrophoresis

1. Samples of DNA are placed in compartments in a soft agar gel and subjected to an electrical current

2. The negative charge on the phosphate groups cause the DNA to move toward the positive pole on the gel

3. The rate of movement of DNA through the gel is based on the size of the fragments

4. Positions of DNA fragments are determined by staining the DNA fragments in the gel

What are the benefits of distinctive electrophoresis patterns?

1. Useful in characterizing DNA fragments

2. Allow for comparison of genetic similarities among samples as in a genetic fingerprint

What is the function of PCR?

Rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques

What are the abilities of PCR?

• can replicate a target DNA from a few copies to billions of copies in a few hours

• can detect cancer from a single cell

• can diagnose an infection from a single gene copy

T/F: PCR uses the same events of DNA replication.

True

What are the events of PCR?

• Opening of the double helix

• Using the exposed strands as templates

• Addition of primers

• Action of DNA a polymerase

What are the specialized ingredients used in PCR

primers and DNA polymerases

What are primers

oligonucleotides that indicate where DNA amplification should begin

What are DNA polymerases

• enzymes responsible for the replication of DNA

• each version of DNA polymerase completes a unique portion of the replication process

Why are DNA polymerases that are isolated from thermophilic bacteria used in PCR?

Because of the high temperatures used

What are the three steps of PCR technique

1. denaturation

2. priming

3. extension

Denaturation

1. heat target DNA to 94 C to separate strands

2. Cool target DNA between 50-65 C

3. Strands stay separated

Priming

Add primers that bind to the complementary strand of DNA

Extension

1. Increase temp to 72 C

2. Add DNA polymerase and nucleotides

3. 2 complete strands of DNA are produced

What is the purpose of real-time PCR

to detect products during the reaction instead of at the end

What are the essential roles of PCR?

• gene mapping

• studying genetic defects and cancer

• forensics

• diagnosing infectious diseases

• taxonomy studies

What is recombinant DNA technology used for

• To deliberately remove genetic material from one organism and combining it with that of a different organism

• forms genetic clones

Example of recombinant DNA Technology

Production of drug alpha-2a interferon (Roferon-A):

• Used to treat hairy cell leukemia and Kaposi’s sarcomas

What is cloning

the removal of a selected gene from an animal, plant or microorganism

Process of cloning

1. Gene is inserted into a vector

2. Vector is inserted into a cloning host

3. Gene is translated into the protein product for which it codes

What is a vector

a plasmid or virus

Plasmid vectors

• Small, well characterized, easy to manipulate

• Can be transferred into appropriate host cells through transformation

• Carry genetic markers for resistance to antibiotics

Virus (bacteriophage) Vectors

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

What are the three important features of cloning vectors

1. an origin of replication (ORI)

   1. needed to be replicated by DNA polymerase of cloning host

2. must accept DNA of the desired size

3. contains a gene that gives/maintains drug resistance

What is a cloning host

bacteria or yeast

What are some desirable features in cloning host (7)

1. Rapid turnover; fast growth rate

2. Can be grown in large quantities using ordinary culture methods

3. Nonpathogenic

4. Genome that is well mapped

5. Capable of accepting plasmid or bacteriophage vectors

6. Maintains foreign genes through multiple generations

7. Will secrete a high yield of proteins from expressed foreign genes

What are the ingredients needed for gene cloning

the target gene and cloning vector

Steps in gene cloning

1. Prepare the isolated gene for splicing into plasmid

   1. Digest gene and plasmid with same restriction enzyme

   2. Results in complementary sicky ends

   3. Ligase seals gene and plasmid together

2. Introduce plasmid to cloning host via transformation

3. Search for recombinant clones

What are some uses of recombinant DNA technology

• Recombinant organisms

• Sources of protein products

• Nucleotide sequences

• Medications that cannot be manufactured by other means

• Large-scale manufacture of hormones and enzymes:

• Insulin

• Human growth hormone

Interferons

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

Interleukins

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

Tumor necrosis factor (TNF)

used to treat cancer

Erthyropoietin (EPO)

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

Human Growth Hormone (HGH)

stimulates growth in children with dwarfism; prevents wasting syndrome

Transgenic Organisms

• AKA GMOs

• Recombinant organisms produced through the introduction of foreign genes

Uses of recombinant microbes

• Prevent ice crystals from forming

• Destroy invading insects

• Make plants more resistant to insect pests

What is the controversy surrounding the release of genetically engineered plants

Concern that transgenic plants will share their genes for herbicide, pesticide, and virus resistance with natural plants leading to “superweeds”

What can an analysis of DNA do?

• reveal genetic abnormalities, ancestry, etc

• determine if one sample of DNA is the same as another sample

What is DNA profiling used for?

• used to differentiate between organisms that might be causing a disease outbreak

• used in criminal investigations to match a suspect to the DNA left at a crime scene