Genetics - Lecture 16: Molecular Genetics Analysis and Biotechnology

How specific DNA fragments can be amplified

1. Gene cloning (in vivo)

2. Polymerase chain reaction (in vitro)

Gene cloning

insertion of DNA fragments into bacteria in a way that makes the fragments stabilize, allowing to be copied by the bacteria they’re in

Polymerase chain reaction (PCR)

Method of enigmatically amplifying DNA fragments

• uses taq polymerase

• uses many components

• Can be used as a reverse technique

Taq polymerase

A DNA polymerase commonly used in PCR reactions

• isolated from bacterium Thermus aquatics, the enzyme is stable at high temperatures, so its not denatures during the strand separation step of the cycle

Reverse-transcription PCR

Technique that amplifies sequences corresponding to RNA

• Reverse transcriptase is used to convert RNA in complementary DNA, which can then be amplified to the usual polymerase chain reaction

Components of PCR

• DNA template

• DNA polymerase

• Primers

• Deoxynucleotide triphosphate (dNTPs)\Magnesium ion (Mg2+)

• Buffer

• Water

3 steps of the PCR

1. Denaturation

2. Annealing

3. Extension

• all steps are repeated, and each time the amount of target DNA is repeated

Denaturation

• at 90-100 C

• Heat the reaction to separate, or denature, the DNA strands

• provides the single-stranded template for the next step

Annealing

• 30-65 C

• Cool the reaction so the primers can bind to their complementary sequenced on the single-stranded DNA

Extension

• 72 C

• Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA

Many different types of PCR

• PCR

• qPCR

• RT-PCR

• qRT-PCR

Real-Time PCR (qPCR)

• modification of the PCR that is used to measure ythe starting amount of nucleic acid

• The amount of DNA amplifies is measures as the reaction proceeds

• Commonly used with RT-PCR to measure gene expression (qRT-PCR)

Reverse-transcription polymerase chain reaction (RT-PCR)

• Technique that amplifies sequenced corresponding to RNA

• Reverse transcriptase is used to convert RNA into complementary DNA, which can then be amplified by the usual polymerase chain reaction

reverse-transcription quantitative real-time polymerase chain reaction (qRT-PCR)

Technique which combines RT-PCR with qPCR to enable the measurement of RNA levels through the use of cDNA in a qPCR reaction, thus allowing detection of gene expression changes

Gene cloning

Insertion of DNA fragments into bacteria in such way that the fragments will be stable and will be copied by bacteria

• uses plasmid vectors

• transformation of host cells with plasmids

• Screening cells for recombinant plasmids (selective markers can confirm which cells have been transformed)

Cloning Vector

Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell

3 important characteristics of cloning vectors

1. an origin of replication

2. Selectable markers

3. 1 or more unique restriction sites

Different types of cloning vectors

• plasmids

• cosmids

• bacterial artificial chromosomes (BACs)

• yeast artificial chromosomes (YACS)

• Ti plasmids

Plasmid vectors

Plasmids

• small, usually circular DNA molecule that is distinct from the bacterial chromosome

• Capable of replicating independently of the bacterial chromosomes

• Insert foreign DNA into plasmid using restriction enzymes

• Linkers are used

Linkers

• Small synthetic DNA fragments that contain one or more restriction sites

• can be attached to the ends of any piece of DNA and used to insert it into a vector site

Transformation (simplified)

bacterial cell → double stranded recombinant plasmid DNA introduced into bacterial cell → cell culture produced hundreds of millions of new bacteria → many copied of purified plasmid isolated from lysed bacteria

Transformation methods

1. Chemical transformation

2. Electroporation

LacZ gene

• can be used to screen for bacteria containing recombinant plasmids

• An artificially constricted plasmid carries a fragment of the lacZ gene and an ampicillin-resistence gene

Expression vector

• cloning vector

• contains DNA sequences (promoter, ribosome binding-site, transcription initiation and termination sites) that allow DNA fragments inserted into the vector to be transcribed and translated

• Purpose is to introduce a specific gene into a host cell (bacteria or yeast) and direct that cell to produce large amounts of the proteins (or RNA) encoded by the gene

To ensure transcription and translation…

A foreign gene may be inserted into an expression vector

Ex: E. coli expression vector has been used

Molecular techniques used to find genes of interest

1. DNA library

2. Genomic Library

3. cDNA (complementary DNA) Library

DNA Library

Collection of cloned (of genes) containing all the DNA fragments from one source

Genomic Library

Collection of bacterial colonies or phages containing DNA fragments that constitute the entire genome of an organism

cDNA Library

Collection of bacterial colonies or phages contains DNA fragments that have been produced by reverse transcription of cellular mRNA

• required for eukaryotic genes that will be expressesed in prokaryotic cells

Ways to determine DNA sequences

• Dideoxy (sanger) sequencing

• Next generation sequencing technologies

• Third generation sequencing technologies or single-molecule real-time (SMRT) sequencing

• DNA fingerprinting

DNA sequencing

Technique used to determine yhe sequence of bases along a DNA molecule

Dideoxy (Sanger) sequencing

• based on replication

• The fragment to be sequenced is used as a template to make a series of new DNA molecules

Next generation sequencing technologies

• Capabale of simultaneously determining the sequenced of many DNA fragments

• These technologies are much faster and less expensive than Sanger dideoxy sequence method (illumination sequencing)

Third generation sequencing technologies or single-molecule real-time (SMRT) sequencing

• determined the sequence of single molecules of DNA or RNA

• allow longer much longer fragments to be sequenced (produce longer reads), which amplifies the assembly of fragments into a complete genome

DNA fingerprinting

• Profiling (used to identify people)

• Technique used to identify individuals by examining heir DNA sequences

The dideoxy-sequencinb reaction requires either special substrate…

• Structure of deoxyribonucleoside triphosphate, the normal substrate for DNA synthesis (dNTP)

• Structure of dideoxyribonucleoside triphosphate, which lacks an OH group on the 3′-carbon atom (ddNTP)

Micro satellites or Short Tandem Repeats (STRs)

• Very short DNA sequence repeated in tandem

• Detected PCR

• Fragments are represented as peaks on a graph

• Homozygous for an STR allele have have a single tall peak

• Heterozygous have two shorter peaks

A DNA profile…

Represents the pattern of DNA fragments produced by performing PCR on the STR loci

Forward Genetics

traditional approach to the study gene function that begins with mutant phenotype and proceeds to a gene that encodes the phenotype

phenotype → genotype

Reverse Genetics

A molecular approach to study of gene function that begins with a genotype (a DNA sequence) and proceeds to the phenotype by altering the sequence or by inhibiting its expression

genotype → phenotype

Transgenic

An organism permanently altered by the addition of a DNA sequence to its genome

Transgene

Foreign gene or other DNA fragment carried by transgenic animal

Transgenic animal…

have genomes that have been permanently altered through recombinant DNA technology