BIOL 3001 Final

DNA

double helix molecule made up of nucleotides, which are the building blocks of genetic information. The sequence of these nucleotides encodes the instructions for building and maintaining an organism.

Genome

encompasses all the DNA within an organism, including all its genes and the non-coding DNA that supports them. It's like the complete instruction manual for an organism.

Human Genome Prokect

a massive, international scientific endeavor to map and sequence the entire human genome. It aimed to identify all the genes within human DNA and make the resulting data publicly available for research purposes.

Bioinformatics

an interdisciplinary field that combines biology, computer science, and information technology to analyze and interpret biological data

non-coding DNA

the portion of an organism's DNA that does not code for proteins. While historically considered inactive or insignificant, it's now understood to play crucial roles in regulating gene expression and other cellular processes.

Sequence inspection

Can be used to locate genes because genes are not simply a random series of nucleotides but instead have distinctive features

Not a foolproof way of locating genes, but still a powerful tool and is usually the first method that is applied to analysis of a new genome sequence

ORF scanning

Effective way of locating most, but not all of the genes in the DNA sequence

Analysis of bacterial sequences is simplified = don't have non-coding introns

Intergenic

stretches of DNA sequences located between genes. These regions can be involved in regulating gene expression and may contain functional elements or "junk DNA". They are also referred to as spacer DNA

Codon bias

not all codons are used equally frequently in the genes of a particular organism

Exon-intron boundaries

Semi-distinct sequence features

Upstream regulatory sequences

DNA sequences located before (or upstream of) a gene's coding region that control the gene's expression

CpG islands

a region of DNA with a high frequency of CpG dinucleotides (cytosine followed by guanine, linked by a phosphate group). These islands are typically found near the promoter regions of genes and play a crucial role in regulating gene expression

GenBank

A database of previously sequenced and identified genes available through the NCBI

designed to provide and encourage access within the scientific community to the most up-to-date and comprehensive DNA sequence information

cDNA

Complementary DNA

DNA copy of an mRNA molecule

Used in GenBank to represent the coding sequences of genes, particularly when studying gene expression or when cloning eukaryotic genes into prokaryotic systems

Lacks introns and only includes expressed regions of a gene

Reverse transcriptase

an enzyme that synthesizes DNA from an RNA template, a process called reverse transcription

cDNA library

A limited gene library using complementary DNA. The library includes only the genes that were transcribed in the cells examined.

reflects the gene expression profile of a specific cell or tissue

Assembly

taking the large numbers of generated DNA sequences and finding areas of overlap between them

De Novo Sequencing

sequencing a novel genome where there is no reference sequence available for alignment.

Fragmenting

Original DNA is broken into smaller pieces, often randomly

Sequencing

Fragments are sequenced, generating a large number of short DNA sequences ("reads")

Alignment and merging

The reads are aligned and merged based on the overlapping sequences to create larger, contiguous sequences called contigs

Scaffolding and ordering

Contigs are ordered and oriented using additional information, and then potentially ordering the scaffolds into chromosomes

Annotation

The assembled sequence is annotated, identifying genes and other functional elements

Gibson assembly

a molecular cloning method used to join multiple DNA fragments together in a single, isothermal reaction

powerful technique known for its efficiency, speed, and ability to assemble large DNA fragments

Needs three enzymes: 5' exonuclease, a DNA polymerase, and a DNA ligase

Alignment

performed when the new DNA sequence generated is compared to existing DNA sequences to find any similarities or discrepancies between them and then arranged to show these features

Basic Local Alignment Search Tool (BLAST)

refers to a suite of programs used to generate alignments between a nucleotide or protein sequence, referred to as a "query" and nucleotide or protein sequences within a database, referred to as "subject" sequences

Alignment score

Numerical value reflecting the quality of the match

Percent identity

How many of the nucleotides are identical between the query and target sequences

E-value

A statistical measure of how likely it is to find a match of this quality purely by chance

Gene prediction

the process of identifying the regions of genomic DNA that encode genes

Evidence based prediction

Relies on previously gathered evidence regarding specific DNA sequences

TATAA box

thymine and adenine rich region of a gene located in most eukaryotic promoter regions

Located 10 bp upstream of the transcription initiation start site

5' UTR

Helps initiate transcription

3' UTR

Aids in transcription termination

Exons

Contain coding information that will be translated

Introns

Found between exons and are spliced or removed from the transcript before translation

Poly-A tail

Series of adenines are added to the 3' end of the mRNA

Start codon

AUG

Signals where protein synthesis should start

Stop codon

UAA, UAG, UGA

Signal protein synthesis to stop

Genome browser

software tool used to visualize and explore genomic data, including DNA sequences and annotations like genes, regulatory elements, and other features

Electrophoresis

technique commonly used in the laboratory to separate charged macromolecules, such as DNA or protein, according to size

may also be based on molecular weight, length and/or structure (shape)

Migration

Movement of charged molecules through the matrix in electrophoresis

DNA sequencing

the process of determining the order of nucleotides (adenine, guanine, cytosine, and thymine) within a DNA molecule

Chain termination sequencing

works by synthesizing new DNA strands complementary to a template, but the process is terminated when a dideoxynucleotide. (ddNTP) is incorporated instead of a normal deoxynucleotide (dNTP). This creates fragments of varying lengths, which are then separated by gel electrophoresis and analyzed to reveal the DNA sequence.

Sanger sequencing

a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication

DNA template

the DNA fragment that is being sequenced.

single-stranded DNA.

Thousands of copies of each fragment are required.

These copies can be created by cloning but most methods now use template DNA fragments produced using the Polymerase Chain Reaction.

This technique allows a researcher to target very specific DNA locations within the genome and amplify millions of near-perfect copies within a few minutes or hours.

DNA primer

a short strand of DNA that binds to the DNA template near the beginning of the fragment being sequenced

Deoxynucleotides

All four of the normal deoxynucleotides are included in the DNA synthesis reactions. The deoxynucleotide triphosphates are abbreviated dATP, dCTP, dGTP and dTTP, depending on which of the four different nitrogenous bases are attached (adenine, cytosine, guanine or thymine). When a nucleotide is added to a growing strand, two phosphates are released. The energy from this release drives the formation of the phosphodiester bond between the incoming nucleotide and the primer end.

DNA polymerase

required to catalyze formation the phosphodiester bonds. Polymerases used in sequencing are usually modified to operate efficiently in test tube environments and to have excellent proofreading capabilities (error prevention)

Reaction buffer

various chemicals and salts are added to highly purified water to mimic conditions in the cell where Replication normally occurs.

Di-deoxynucleotides

identical to the deoxynucleotides normally used during DNA Replication, with the exception that they lack the OH group on the 3' position of the ribose sugar.

During DNA sequencing, a di-deoxynucleotide can be added to a growing DNA strand but because the 3' OH is missing, the next phosphodiester bond cannot form (i.e. chain termination occurs).

Where should I begin my Sanger sequencing read?

Because the smallest fragments travel further in the gel than larger fragments, the DNA fragment at the very bottom of the gel is where you should begin. This fragment is a single "faulty" nucleotide attached to the primer you used in the sequencing reaction. IF the primer were 10 bases in length, the fragment at the bottom of the gel would be 11 bases long.

Which base am I looking at on the DNA fragment in sanger sequencing?

Remember, you ran FOUR different reactions, each containing a single and different di-deoxynucleotide. Those are the samples loaded in each lane. Read the nucleotide at the top of gel to know which one was added.

Chromatography

a laboratory technique used to separate the components of a mixture.

Chromatogram

a visual representation of the separation of components (DNA) in a mixture achieved through chromatography. It is essentially a graph where the signal intensity from a detector is plotted against time (or elution volume). Each peak on the chromatogram corresponds to a different component of the mixture, in this case, a different strand of DNA.

Genotyping

the process of determining the specific genetic makeup, or genotype, of an individual.

Microsatellite genotyping

a molecular biology technique used to identify and analyze variations within microsatellites, also known as short tandem repeats (STRs) or simple sequence repeats (SSRs), in DNA

Polymorphic

the state or quality of having or existing in multiple forms; the microsatellite region across individuals in a population will vary significantly

Linkage maps

a representation of the relative positions of genes or genetic markers on a chromosome, based on how frequently they are inherited together

Forward and reverse primer

Short, single stranded DNA sequences that bind to specific regions on a DNA template, flanking the target DNA sequence to be amplified. The forward primer binds to the antisense (or template) strand of DNA, while the reverse primer binds to the sense (or non-template) strand

Homozygotes appear as

a SINGLE band

Heterozygotes appear as

two bands

DNA profiling

the process where a specific DNA pattern, called a profile, is obtained from a person or sample of bodily tissue

Karyogram

shows the chromosomes of an organism in homologous pairs of decreasing length.

Genic DNA

~20% of the total genome

composed of the actual "coding regions" of DNA, those regions that provide the blueprint and information to synthesis proteins in the cells of the body

Also comprised of all the regulatory regions of the DNA that controls the synthesis of proteins. Regions of DNA like promoters, enhancers, repressors or polyadenylation signals are some examples of regulatory regions.

Extragenic DNA

~80% of the total genome

has non-coding regions of DNA. In the past, it has been classified as "Junk DNA" and thought to have no functionality

About half (~50%) of extragenic DNA is composed of what is termed "Repetitive DNA". A simple definition of this type of DNA are sequences that are present in multiple copies within a genome

Tandem repeats

sequences of DNA where a single unit of one or more base pairs is repeated multiple times in a tandem or head-to-tail fashion. These include all the "satellite regions" of the genome (minisatellites, microsatellites, alpha satellites, etc.).

Interspersed repeats

sequences of DNA that are repetitive in nature but are scattered throughout the genome rather than being clustered together like satellite regions. These include elements like interspersed repeats, long terminal repeats and transposons.

Polymorphic regions

some specific regions that are highly variable between individuals

Short Tandem Repeats (STRs)

used to create a DNA profile that is highly unique to each individual person.

Aka microsatellites

Regions of non-coding DNA that contain repeats of the same nucleotide sequence

Electropherograms

A graphical representation of STRs located on a person's chromosomes used to identify and tie suspects to a crime scene

Female (pedigree)

Male (pedigree)

Affected individuals

Carriers

Dead (pedigree)

Gender not specified

Spontaneous abortion (miscarriage)

Still born

Terminated pregnancy

dizygotic twins (fraternal)

Monozygotic twins (identical)

Mating

Consanguinous mating (inbreeding)

Extramarital relationship

proband

Pregnancy

Ectopic pregnancy

Unaffected individual

Cannot have any alleles of a dominant trait (because a single allele of a dominant trait causes an individual to be affected)

Patterns that indicate a recessive trait

If any affected has 2 unaffected parents

If the trait is autosomal, both parents can be unaffected carriers of the disease

If the trait is x-linked, the son must have inherited his allele from his mother only, and his father can be unaffected

Autosomal recessive

If any affected founding daughter has 2 unaffected parents

Both parents must have an allele

A trait that skips a generation is a classic clue

May appear, disappear, and reappear across generations

Male/females equally affected

Unaffected mating having an affected offspring (both parents are heterozygous)

X-linked recessive

When an affected non-founding son has 2 unaffected parents the disease must be X-linked recessive

A male cannot be affected by a single autosomal recessive allele, but can be affected by a single X-linked recessive allele

More males than females affected, males need only one copy of the defective allele (hemizygous)

If no females (or only a few) are affected, it is likely that the trait is X-linked

Affected sons receive the allele on the X from their mothers

An affected female must have an affected father

Patterns that indicate a DOMINANT trait

The disease must be DOMINANT if every affected child of NON-FOUNDING parents has an affected parent.

No child could be affected by a single autosomal recessive allele, or X-linked recessive allele, so the trait is dominant.

Autosomal dominant

When affected son of non-founding parents has an affected father

A father does not transmit X-linked alleles to a son, so the disease cannot be X-linked dominant

When an affected daughter of non-founding parents has an affected father, we cannot determine whether the dominant disease is autosomal or x-linked. The father can transmit either an autosomal dominant allele, or an X-linked dominant allele to his daughter

Usually found in every generation

Males/females equally affected

An affected individual must have at least one affected parent

Affected individual usually has affected offspring

X-linked dominant

More females than males affected, females have 2 chances to receive allele (XX)

An affected individual must have at least one affected parent

Trait observed every generation (cannot skip a generation)

Cancer

Uncontrolled cell growth

Invasion

the spread of cancer cells from the original tumor to other parts of the body

Metastisis

the process by which cancer cells spread into nearby tissues

Angiogenesis

the process of new blood vessels forming from existing blood vessels

Green boxes

start codons