Genetics Chapter 20 and 21

What is recombinant DNA?

produced by artificially joining DNA from multiple biological sources to create DNA sequences not found together in nature

What are the two tools of recombinant DNA technology?

Restriction Enzymes and DNA Cloning Vectors

What is Recombinant DNA Technology used for?

- to isolate, replicate, and analyze genes

- to create genetic constructs to drive gene expression in a host cell

What is molecular cloning?

A set of techniques used to assemble recombinant DNA molecules and direct their replication

What are the two basic steps of Molecular Cloning?

- Insert DNA fragments into cloning vectors to make a recombinant DNA molecule

- Transport recombinant DNA into a living cell to be copied.

What are Cloning Vectors?

small pieces of DNA that a foreign DNA fragment can be inserted into, which can then be placed into cells for the purposes of molecular cloning

What are the characteristics of a cloning vector?

- Can be stably maintained in an organism and replicate cloned DNA fragments in a host cell

- Can be of viral, bacterial, or eukaryotic origin

- Must be able to replicate inside the host

What is a common cloning vector?

plasmid

Define plasmid.

Extrachromosomal, circular double-stranded DNA molecule from bacteria

What are the three main features of plasmid vectors?

- origin of replication to allow for their replication in a host cell

- restriction enzyme cut sites to allow insertion of DNA fragments

- carry a selectable gene marker to distinguish host cells that have taken them up from those that have not

What are Multiple Cloning Sites?

- Restriction sites for commonly used restriction enzymes

- Allow scientists to clone a range of different fragments

What happens to sequences that are not needed?

removed and replaced with unique restriction sites for insertion of cloned DNA.

What are Restriction Enzymes?

bind to DNA at specific recognition sequence and cleaves DNA to produce restriction fragments

Most restriction sites are ______.

palindromes

Restrictive enzymes cleave both or one strand of DNA?

both

What are sticky ends?

Cohesive ends produced with overhangs?

What are blunt ends?

Fragments produced with double-stranded ends

Define DNA Ligase?

Joins restriction fragments of DNA to form a double helix together by forming a covalent bond and sealing the nicks in the phosphodiester backbone

How are plasmids introduced inot bacteria?

via transformation

What are BACs?

Bacterial artificial chromosomes

What are YACs?

yeast artificial chromosomes

What are Expression Vectors?

Designed to ensure mRNA expression of cloned gene and to produce large quantities of encoded protein in host cell

Are Expression vectors available in both prokaryotes and eukaryote host cells?

yes

How is recombinant DNA made using restriction enzymes and host cells?

1. Plasmid DNA and DNA to be cloned are cut with same restriction enzyme

2. DNA restriction fragments from DNA to be cloned are added to the linearized vector in the presence of DNA ligase

3. Recombinant DNA is produced and introduced into bacterial host cells by transformation

What does cDNA stand for?

Complementary DNA

What is cDNA library?

Contains complementary DNA copies made from mRNAs present in the cell population, representing genes actively transcribed at the time cells were collected for mRNA isolation

How is cDNA library constructed?

- Isolating mRNA from cells

- Synthesizing complementary DNA using reverse transcriptases

- Cloning cDNA molecules into vector

What is the function of DNA sequencing?

Determine the nucleotide sequence of fragment of DNA in your library

How can DNA sequencing be used?

- to sequence a genome or determine mutations (from genomic DNA library)

- to determine which genes are expressed, and in what quantities

What does PCR stand for?

Polymerase Chain Reaction

What is the function of PCR?

copies specific DNA sequence via in vitro DNA synthesis reactions, amplifying target DNA sequences present in very small quantities

What does PCR require?

- Template DNA

- Primers

- DNA polymerase

- dNTPs (nucleotide monomers)

- Appropriate salt and buffer conditions

What are the two primers PCR requires?

forward and reverse

Define primers.

Short, single-stranded sequences, one complementary to 5′ end and another complementary to 3′ end

Primers _____ to denatured DNA.

anneal

What do primers determine?

which part of the template DNA will be amplified

What are the three steps of PCR?

1. denaturation

2. annealing

3. extension

Define denaturation.

high temp to break hydrogen bonds and denature double-stranded DNA to make singe-stranded

Define annealing.

lower temp to allow primers to bind/hybridize/anneal to template DNA

Define extension.

optimal temperature for DNA polymerase (heat-stable DNA polymerase)

What is used to amplify DNA exponentially?

thermocycler

- new and old strands serve as templates

What are the limitations of PCR?

- Some info about nucleotide sequence of target DNA is required in order to design primers

- Minor contamination from other sources can cause problems

- cannot amplify very long segments of DNA

What are some applications of PCR?

- molecular cloning

- Allows for screening of mutations and DNA variants

- Testing for the presence of microorganisms or viruses

- Preparing DNA for DNA sequencing

What is RT- PCR?

Reverse transcription PCR

What is the function of RT-PCR?

Methodology for studying gene expression (to help analyze mRNA production by cells or tissues)

- Make DNA copies from an RNA molecule

What is used in combination with RT-PCR to create cDNA?

reverse transcriptase

What is qPCR?

Quantitative real-time PCR

What is the function of qPCR?

Real-time PCR allows researchers to quantify the amount of template DNA that was present in the sample

How can DNA be analyzed with agarose gel using electrophoresis?

Used to analyze size of DNA fragments

How does DNA fingerprinting relate to PCR?

- PCR used to amplify regions with various numbers of repeats

- Number of repeats will determine size of PCR fragment

- Run PCR reactions on gel and determine which person's DNA matches the unknown DNA sample

How can DNA sequencing be used to analyze DNA?

allows you to determine the nucleotide sequence

What is DNA similar to?

a modified in vitro DNA synthesis reaction

What does DNA sequencing need?

- DNA template

- Primer

- DNA polymerase

- Nucleotides (standard dNTPs and modified nucleotides)

What are modified nucleotides?

- ddNTPs (missing the 3' OH so that another nucleotide can't be added, chain termination)

- Labeled with radioactive or fluorescent dye

What does ddNTPs stand for?

Dideoxynucleotides

What is a ddNTP?

Deoxynucleotide with a hydrogen at 3′ instead of an OH

Describe Dideoxynucleotide chain-termination sequencing.

Dideoxynucleotide causes DNA synthesis to terminate, creating different size fragments

What is computer-automated DNA sequencing?

computer-automated Sanger reaction-based technology to generate large amounts of sequence DNA, which enabled rapid progress of Human Genome Project

What do computer-automated DNA sequences use?

fluorescent dye-labeled dideoxynucleotides (ddNTPs)

Why might a scientist want to alter a genome?

- Generate an animal in which specific tissues can be visualized

- Knock out a gene that you think is involved in a disorder

- Knock down a gene that you think may be involved in a biological process

- Rescue a genetic mutation

What can a reporter gene construct be used for?

to label cells that exhibit specific patterns of gene expression

What is transgenesis?

insertion of (usually engineered) DNA into an organism's genome

- transgenic mice

- brainbow mice

Describe brainbow mice.

use multiple combinations of different fluorescent labels to generate many distinctly labeled neurons to decipher neural circuitry of complex brains

What is the concept of Gene targeting?

To manipulate specific alleles, locus, o rbase sequence and learn its function on gene of interest

What is the concept of Gene Knockout?

To disrupt or eliminate specific gene/genesand see "what happens"

How are knockout mice created?

- Construct targeting vector by creating segment of DNA for introduction into cell

- Undergoes homologous recombination with gene of interest and renders it nonfunctional

What is the Cre-lox system used for?

making conditional KOs

What are Conditional KOs?

Allows scientist to control when target gene is disrupted—a particular time in development

What are knock-in animals?

- Express or overexpress particular gene of interest (transgene)

- Vector with transgene undergoes homologous recombination into host genome

- Vector with transgene can also be put into ES cells and injected into embryos

What do targeted mutagenesis techniques rely on?

bringing enzymes that cut DNA to specific sites on the chromosome

What are the three main types of targeted mutagenesis techniques?

Zinc finger nuclease, TALENs, and CRISPR-Cas9

Describe the Cre-lox system.

- Promoter for Cre designed as tissue specific

- Conditional KOs made by inserting sequences called loxP sites

- Sequences are introduced into germ line

What does CRISPR stand for?

clustered regularly interspaced short palindromic repeats

Describe CRISPR-Cas9.

Gene editing method that involves the use of specifically engineered DNA-modifying enzymes (nucleases) that create changes in a specific sequence to remove, correct, or replace a defective gene or parts of a gene

How is gene editing followed through with?

based on using different nucleases to create targeted breaks in the genome in a sequence-specific manner

What is Cas9?

An endonuclease, an enzyme that cuts DNA, that binds to a guide RNA which recruits Cas9 to a precise location in the genome so it can cut DNA at that location, near a PAM site.

What can CRISPR-Cas9 be used for?

- disrupt a sequence

- correct a sequence

- insert a sequence

- regulate expression of a sequence

What is meant by the term "genome"?

Complete set of DNA in a single cell of an organism

What is genomics?

The study of genomes

What are the different kinds of genomics?

- Structural genomics

- Functional genomics

- Comparative genomics

- Metagenomics

What does genomics allow for?

sequencing of entire genomes

What is the most widely used strategy for sequencing and assembling an entire genome?

shotgun cloning

Describe Shotgun Cloning?

1) Genomic DNA is cut into fragments and contigs are made

2) Entire chromosome is assembled by computer program

3) Fragments are aligned based on identical DNA sequences

What are Contigs?

Overlapping fragments adjoining segments that collectively form one continuous DNA molecule within chromosome

What is the purpose of Algorithm-based software programs?

Create a DNA-sequence alignment, a similar sequence lined up for comparison, which identifies overlapping sequences, allowing scientists to reconstruct their order in the chromosome

Describe computer-automated DNA sequencers?

- Designed for high-throughput sequencing

- Made genomics possible

- Essential for Human Genome Project

Define Bioinformatics.

Uses computer-based approaches to organize, share, and analyze data related to:

- Gene structure

- Gene sequence and expression

- Protein structure and function

What is GenBank?

The largest publicly available database of DNA sequences

- Maintained by National Center for Biotechnology Information (NCBI)

What does BLAST stand for?

Basic Local Alignment Search

What is BLAST?

Software application used to compare a segment of genomic DNA to sequences throughout major databases

What is the purpose of BLAST?

Identifies portions that align with or are the same as existing sequences (similarity score)

What are protein-coding regions?

Annotation of sequence reveals several identifiable features indicating that the sequence contains a protein-coding gene

What are some examples of protein-coding regions?

- Promoter sequence

- Initiation sequence

- Exons with open reading frames

What does ORFs stand for?

Open reading frames

What are ORFs?

Sequences of triplet nucleotides translated into amino acid sequence of a protein

What are the characteristics of an ORF?

- no stop codons

- Suggestive of protein-encoding gene

- Typically begin with initiation sequence ATG

What are BLAST searches used for?

to screen databases and compare a DNA sequence to a known sequence

Describe similarity in relation to gene sequences.

A genome sequence statistically similar to gene with known function likely encodes for protein with similar function

What are homologous genes?

Genes that are evolutionarily related

What are orthologs?

Homologous genes in different species thought to have descended from common ancestor