BIOL 3000

gene

- entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide

- includes coding region and all DNA sequences required to synthesize particular RNA transcript

central dogma

DNA --> RNA --> protein

DNA

- deoxyribonucleic acid

- double helix structure composed of two intertwined strands, resembling a twisted ladder

- made of nucleotides with sugar, phosphate, and ntirogenous base

- adenine, guanine, cytosine, and thymine

key components of DNA function

- genetic information

- heredity

- protein synthesis

DNA vs genome

DNA is molecule that carries genetic instructions for all living organisms, while genome is the complete set of DNA within an organism

key differences: scope (genome is larger), function (DNA provides instructions, genome is complete set of instructions), and analogy (dna = lettters, genome = dictionary)

human genome project

massive, international scientific endeavor to map and sequence the entire human genome

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

open reading frames

regions of DNA that are likely to encode a protein

begin with initiation codon (ATG) and ends with termination codon (TAA, TAG, or TGA)

one way of looking for genes

each DNA sequence has six reading frames (three in one direction and three in the reverse)

effective for bacterial genomes, less for eukaryotic

intergenic

stretches of DNA sequences located between genes

codon bias

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

upstream regulatory sequences

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

can be used to locate the regions where genes begin

CpG islands

a region of DNA with a high frequency of CpG dinucleotides

typically found near promoter regions

genbank

NIH genetic sequence database, annotated collection of all publicly available DNA sequences

mostly cDNA sequences

cDNA

complementary DNA, DNA copy of an mRNA molecules

expressed portion of a gene, lacks introns

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

reverse transcriptase

an enzyme that synthesizes DNA from an RNA template

assembly

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

gradually, pieces are put together and sequenced DNA strand will get longer

mostly de novo sequencing

de novo sequencing

organism of interest is being sequenced for the first time

no existing reference genome to use as a template for the assembly of the unknown genome

assembly steps

1. fragmenting - original DNA is broken randomly into smaller pieces

2. sequencing - fragments are then sequenced, generating a large number of short DNA sequences ("reads")

3. alignment and merging - reads are aligned and merged based on overlapping sequences to create larger, contiguous sequences called contigs

4. scaffolding and ordering - further steps can involve scaffolding, where contigs are ordered and oriented using additional information, and then potentially ordering the scaffolds into chromosomes

5. annotation - assembled sequence is annotated, identifying genes and other functional elements

gibson assembly

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

uses 5' exonuclease, DNA polymerase, and DNA ligase

alignment

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

BLAST

basic local alignment search tool

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

finds local regions of high similarity

"creates words": breaks down query sequence into short overlapping segments (words)

searches database: searches database for target sequences that contain these words or very similar ones

extending matches: once match is found, BLAST extends alignment in both directions to see how far similarity extends

alignment score (BLAST)

a 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

annotation

1. gene prediction

2. manual annotation

TATAA box

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

10 bp upstream of transcription initiation start site

5' UTR

untranslated region, plays important role in initiating 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 added to 3' end of mRNA after transcription

start codon

AUG, signals where protein synthesis should start

stop codon

(UAA, UAG, UGA), signal protein synthesis to halt

genome browser

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

T/F: Assembly is used when there is no existing reference genome and Alignment is used to compare DNA to existing sequences.

true

Which of the following is in the correct 5' to 3' order as they would line up on a gene?

Promotor, Exon, Intron, Exon, Terminator

De Novo sequencing refers to which of the following?

sequencing an organism for the first time

T/F: Genes are a random sequence of nucleotides undefined by any distinctive features, and consist of both coding and non-coding DNA regions.

false

T/F: The term cDNA is also used, typically in a Bioinformatics context, to refer to an mRNA transcript's sequence, expressed as DNA bases (deoxy-GCAT) rather than RNA bases (GCAU).

true

The Genome Browser shows how many reading frames for any particular gene?

6

electrophoresis

technique commonly used in biology laboratories to separate biomolecules according to their size

with DNA (negatively charged), DNA will migrate towards positive electrode and be separated by size (shorter lengths move faster)

migration

movement of biomolecules through a matrix or gel

"run to red" - positive electrode is red one

smaller molecules move through matrix more quickly

genotyping

the process of determining differences in the genetic makeup of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence

microsatellites

a tract of repetitive DNA in which certain DNA motifs are repeated, typically 5-50 times

DNA sequencing

process of determining order of nucleotides 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 is incorporated instead of a normal deoxynucleotide; this creates fragments of varying lengths, which are then separated by gel electrophoresis and analyzed to reveal DNA sequence

microsatellite genotyping

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

microsatellite allele - homozygote

one band

microsatellite allele - heterozygote

two bands

Performing electrophoresis in water, instead of buffer:

won't work because the buffer provides ions to allow the current to move

__________ is a series of techniques by which biomolecules can be separated based on size, length and/or shape.

electrophoresis

T/F: Due to its relative negative charge, DNA will tend to move towards the positive end of the gel.

true

It will be very easy to distinguish homozygotes from heterozygotes during this lab exercise. Homozygotes will all show _______ band(s) and heterozygotes will all show __________ band(s).

1, 2

_______________ can compare translated nucleotides to proteins.

BLAST

BLAST stands for _______________________________________.

basic local alignment search tool

When genotyping to determine relationships, we are looking for alleles that _______________?

exclude someone from the potential relationship

T/F: We use genotyping to determine relationships because it is provides indisputable scientific facts.

false

DNA profiling

the analysis of DNA from samples of body tissues or fluids, especially when conducted in order to predict susceptibility to a specific disease

DNA extraction

The cell and nuclear membranes need to be broken up to allow the DNA to be free in solution. Once the DNA is free, it can be separated from all other cellular components. After the DNA has been separated in solution, the remaining cellular debris can then be removed from the solution and discarded, leaving only DNA.

RFLP analysis

DNA is collected from cells and cut into small pieces using a restriction enzyme (a restriction digest). This generates DNA fragments of differing sizes as a consequence of variations between DNA sequences of different individuals. The fragments are then separated on the basis of size using gel electrophoresis. The separated fragments are then transferred on to a nitrocellulose or nylon filter; this procedure is called a Southern blot.

southern blot

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

PCR

The process mimics the biological process of DNA replication, but confines it to specific DNA sequences of interest. With the invention of the PCR technique, DNA profiling took huge strides forward in both discriminating power and the ability to recover information from very small (or degraded) starting samples

STR analysis

Simple sequences or short tandem repeats (STR), uses highly polymorphic regions that have short repeated sequences of DNA (the most common is 4 bases repeated, but there are other lengths in use, including 3 and 5 bases). Because unrelated people almost certainly have different numbers of repeat units, STRs can be used to discriminate between unrelated individuals. These STR loci (locations on a chromosome) are targeted with sequence-specific primers and amplified using PCR. The DNA fragments that result are then separated and detected using electrophoresis. There are two common methods of separation and detection, capillary electrophoresis (CE) and gel electrophoresis.

DNA databases

There are now several DNA databases in existence around the world. Some are private, but most of the largest databases are government-controlled. The United States maintains the largest DNA database, with the Combined DNA Index System (CODIS) holding over 13 million records as of May 2018.

what was the first heritable variation detected in human DNA called?

restriction fragment-length polymorphism

What was the first public use of genetic fingerprinting?

proving a boy was the biological child of his mother

DNA from skeletal remains was able to prove that the remains belongs to Josef Mengele because of what development(s)?

PCR, STR

In forensic cases, what is sometimes typed because samples contain many more copies of this than what is normally typed?

mitochondrial DNA

creating a DNA profile using STRs

1. obtain sample

2. isolate DNA

3. run PCR to amplify DNA

4. determine size of STRs

5. determine if a match exists

pedigree assumptions

1. complete penetrance

2. rare-in-population

3. not y-linked

pedigree

a chart that shows the presence or absence of a trait within a family across generations

complete penetrance

the gene or genes for a trait are expressed in all the population who have the genes

incomplete penetrance

the gene or genes for a trait that is expressed in only part of the population who have the genes

female

male

affected individuals

carriers

dead

gender not specified

spontaneous abortion (miscarriage)

still born

terminated pregnancy

dizygotic twins (fraternal)

monozygotic twins (identical)

mating

consanguineous mating

extramarital relationship

pedigree clues

1. unaffected individual cannot have any alleles of a dominant trait

2. individuals marrying into the family are assumed to have no disease alleles

3. unaffected individual can be a carrier of a recessive trait

4. when a trait is x-linked, a single recessive allele is sufficient for a male to be affected

5. a father transmits his allele of x-linked genes to his daughters, but not his sons. a mother transmits an allele of x-linked genes to both her daughters and her sons

autosomal recessive

- if any affected founding daughter has 2 unaffected parents the disease must be autosomal recessive

- an affected individual must inherit a recessive allele from both parents, so both parents must have an allele

- if the father had an x-linked recessive allele, he would have to be affected

recessive

- if an affected founding son has 2 unaffected parents, we cannot determine if the recessive disease is autosomal or x-linked

- 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

x-linked recessive

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

- the father, who is marrying in, does not have any disease alleles; so affected son inherits an allele only from his unaffected mother

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

dominant trait

- must be dominant if every affected child of non-founding parents has an affected parent

- the unaffected mother, who is marrying in, does not carry an allele for the disease; so the affected child inherits an allele only from the affected father

- no child could be affected by a single autosomal recessive allele, or x-linked recessive allele, so the trait is dominant

autosomal dominant

when an affected son of non-founding parents has an affected father the disease must be autosomal dominant

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

autosomal recessive trait

- trait skips generation is clue

- males/females equally affected

- unaffected mating having an affected offspring (heterozygous parents)

autosomal dominant trait

- usually found in every generation

- males/females equally affected

- affected individual must have at least one affected parent

- affected individual usually has affected offspring

x-linked recessive trait

- more males than females affected (males only need one copy)

- if no or few females are affected, likely x-linked

- affected sons receive the allele on the X from their mothers

- an affected female must have an affected father

x-linked dominant trait

- more females than males affected, females have 2 chances to receive allele

- an affected individual must have at least one affected parent

- trait observed every generation (cannot skip a generation)

If a population of people had the exact same genes for a trait but only some people exhibit the trait we would say the trait has:

incomplete penetrance

One way to distinguish dominant and recessive traits in a pedigree is that the former will not skip generations but the latter will. But will recessive traits always skip generations?

no, if both parents are homozygous for the recessive trait it will not skip generations